US6376459B1 - Inhibiting B cell activation with soluble CD40 or fusion proteins thereof - Google Patents

Inhibiting B cell activation with soluble CD40 or fusion proteins thereof Download PDF

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US6376459B1
US6376459B1 US08/114,944 US11494493A US6376459B1 US 6376459 B1 US6376459 B1 US 6376459B1 US 11494493 A US11494493 A US 11494493A US 6376459 B1 US6376459 B1 US 6376459B1
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cd40cr
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Alejandro A. Aruffo
Jeffrey A. Ledbetter
Ivan Stamenkovic
Randolph Noelle
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Bristol Myers Squibb Co
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Definitions

  • the present invention relates to a counter-receptor, termed CD40CR, (also known as CD40 ligand) for the CD40 B-cell antigen, and to soluble ligands for this receptor, including fusion molecules comprising at least a portion of CD40 protein. It is based, at least in part, on the discovery that a soluble CD40/immunoglobulin fusion protein or antibody specific for gp39 on T cells was able to inhibit helper T-cell mediated B-cell activation by binding to a novel 39 kD protein receptor on helper T-cell membranes.
  • CD40CR also known as CD40 ligand
  • the present invention provides for a substantially purified CD40CR receptor; for soluble ligands of CD40CR, including antibodies as well as fusion molecules comprising at least a portion of CD40 protein; and for methods of controlling B-cell activation which may be especially useful in the treatment of allergy or autoimmune disease, including graft-versus-host disease (GVHD) and rheumatoid arthritis.
  • GVHD graft-versus-host disease
  • T h formed physical conjugates with class II major histocompatibility complex (MHC) compatible, antigen-presenting B-cells (Vitetta et al., (1987) Immunol. Rev. 99:193-239) and that it was the B-cells within these conjugates that responded to T h (Bartlett et al., (1989) J. Immunol. 143:1745-1754).
  • MHC major histocompatibility complex
  • T h -derived lymphokines exerted potent growth and differentiative effects on B-cells
  • soluble factor(s) released in proximity by activated T h mediated the activation of the interacting B-cell.
  • none of the molecularly cloned lymphokines, alone or in combination manifested the ability to induce B-cell cycle entry.
  • plasma membrane fractions from activated T h induced B-cell cycle entry Hodgkin et al., (1990) J. Immunol. 145:2025-2034; Noelle et al., (1991) J. Immunol. 146:1118-1124).
  • PM Act Purified plasma membranes from activated T h (PM Act ) have been used to investigate the nature of this effector function (Hodgkin et al. (1990) J. Immunol. 145:2025-2034; Noelle et al., (1991) J. Immunol. 146:1118-1124).
  • PM Act required 4-6 hours of activation, de novo RNA synthesis and was protein in nature (Bartlett et al., (1990) J. Immunol. 145:3956-3962).
  • the present invention relates to a counter-receptor, termed CD40CR, for the CD40 B-cell antigen, and to soluble ligands for this receptor, including fusion molecules comprising at least a portion of CD40 protein. It is based, at least in part, on the discovery that a soluble CD40/immunoglobulin fusion protein was able to inhibit helper T-cell mediated B-cell activation by binding to a novel 39 kD receptor protein (termed “CD40CR” for CD40 counter-receptor) on helper T-cell membranes, and on the discovery that a monoclonal antibody, termed MR1, directed toward this 39 kD receptor was able to inhibit helper T-cell mediated activation of B-cells.
  • CD40CR a counter-receptor
  • the present invention provides for a substantially purified CD40CR receptor; for soluble ligands of CD40CR, including antibodies as well as fusion molecules comprising at least a portion of CD40 protein; and for methods of controlling B-cell activation.
  • B-cell activation in a subject may be inhibited by contacting helper T cells of the subject with effective amounts of a soluble ligand of CD40CR.
  • Such inhibition of B-cell activation by interfering with CD40CR of helper T cells may be especially useful in the treatment of allergy or autoimmune disease, including, in particular embodiments, GVHD and rheumatoid arthritis.
  • One advantage of the present invention is that it enables immune intervention in an aspect of the immune response which is not antigen specific.
  • Many current therapies for allergy include desensitization to particular antigens, and require that each patient be tested in order to identify antigens associated with sensitivity.
  • exhaustive analysis of a patient's response to each and every potential allergen is virtually impossible.
  • the causative antigen is, generally, unknown or even irrelevant to the disease process.
  • the present invention which relates to the antigen nonspecific CD40/CD40CR interaction, circumvents the need to characterize the antigen associated with allergy or autoimmunity.
  • the present invention may be used to particular advantage in the treatment of allergic conditions in which the immunogen is not known, or has multiple components, for example, in hay fever or in procainamide induced lupus. It may also be useful in acute treatment of immune activation, for example, in therapy for anaphylaxis.
  • FIGS. 1A-1C Effect of monoclonal antibodies and CD40-Ig on the induction of B-cell RNA synthesis by PM Act .
  • FIG. 1 A Resting B-cells were cultured with PM rest or PM Act from T h 1. 25 ⁇ g/ml of anti-CD4, anti-LFA-1 or anti-ICAM-1 or a combination of each of these (each at 25 ⁇ g/ml) was added to wells containing PM Act , and B-cell RNA synthesis was measured by incorporation of [ 3 H]-uridine. B-cell RNA synthesis was assessed from 42 to 48 hours post-culture. Results presented are the arithmetic means of triplicate cultures+/ ⁇ s.d., and are representative of 5 such experiments.
  • FIG. 1 B Resting B-cells were cultured with PM Act from T h 1 ( ⁇ , ⁇ ) or T h 2 ( ⁇ ). To the T h 1 PM Act containing cultures ( ⁇ , ⁇ ), increasing amounts of CD40-Ig ( ⁇ ) or control protein CD7E-Ig( ⁇ ) were added. To the T h 2 PM Act containing culture ( ⁇ ), increasing amounts of CD40-Ig were added. B-cell RNA synthesis was assessed from 42 to 48 hours post-culture. Results presented are the arithmetic means of triplicate cultures+/ ⁇ s.d., and are representative of 3 such experiments.
  • FIG. 1 C Resting B-cells were cultured with LPS (50 ⁇ g/ml) or PM Act . To cultures, CD40-Ig (25 ⁇ g/ml; hatched) or CD7E-Ig (25 ⁇ g/ml; solid) were added. RNA synthesis was determined as described in FIG. 1 A. Results presented are the arithmetic mean of triplicate cultures+/ ⁇ s.d., and are representative of 3 such experiments.
  • FIGS. 2A-2B CD40-Ig inhibited B-cell differentiation and proliferation.
  • FIG 2 A Resting B-cells were cultured with PM Act , rIL4 (10 ng/ml) and rIL5 (5 ng/ml). Either at the initiation of culture, or on days 1, 2 or 3 post-initiation of culture, CD40-Ig or CD7E-Ig (25 ⁇ g/ml) were added. On day six of culture, SN from individual wells were harvested and quantitated for IgM( ⁇ ) and IgG,( ⁇ ) using an anti-isotype specific ELISA, as described in (Noelle et al., (1991) J. Immunol. 146:1118-1124).
  • IL4 and IL5 In the presence of PM Act , IL4 and IL5, (in the absence of added CD40-Ig) the concentrations of IgM and IgG 1 were 4.6 ⁇ g/ml and 126 ng/ml, respectively. Cultures which received CD7E-Ig (25 ⁇ g/ml) on Day 0 produced 2.4 ⁇ g/ml and 89 ng/ml of IgM and IgG 1 , respectively. In the absence of IL4 and IL5, no IgM or IgG 1 was detected. Results are representative of 3 such experiments.
  • FIG. 2B T h 1 were rested or activated with anti-CD3 for 16 hours, irradiated and cultured ( 1 ⁇ 10 4 /well) with resting B-cells (4 ⁇ 10 4 /culture) in the presence of IL4 (10 ng/ml). Between 0 and 25 ⁇ g/ml of CD40-Ig ( ⁇ ) or CD7E-Ig ( ⁇ ) were added to cultures. From 66-72 hours post-culture, wells were pulsed with 1.0 ⁇ Ci of [ 3 H]-thymidine and harvested. The dotted line indicates the response of B-cells to resting T h . Results presented are the arithmetic mean of triplicate cultures+/ ⁇ s.d., and are representative of 2 such experiments.
  • FIGS. 3A-3B CD40-Ig detected a molecule expressed on activated, but not resting T h .
  • Resting (FIG. 3A) and activated (FIG. 3B) T h were harvested and incubated with fusion proteins for 20 minutes at 4° C., followed by FITC-conjugated goat anti-hIgG (25 ⁇ g/ml). Percentage positive cells and MFI were determined by analysis of at least 5000 cells/sample. Results are representative of 6 such experiments.
  • CD40-Ig binding is indicated by a filled-in profile.
  • FIG. 4 CD40-Ig immunoprecipitated a 39 kD protein from lysate of activated T h 1.
  • T h 1 were rested or activated with insolubilized anti-CD3 for 16 hours.
  • [ 35 S]-labelled proteins from resting or activated T h were immunoprecipitated with purified antibodies or fusion proteins (1-10 ⁇ g). The gel profile is representative of 3 such experiments.
  • FIGS. 5A-5B MR1 and CD40-Ig recognized the same molecule expressed on activated T h .
  • FIG. 5 A Activated T h were fluorescently stained with MR1 or control Ig.
  • graded concentrations of MR1 or control hamster Ig anti- ⁇ / ⁇ TCR
  • anti-CD40 20 ⁇ g/ml
  • FIG. 5 B Proteins from [ 35 S]-methionine-labelled, activated T h were immunoprecipitated with MR1 (10 ⁇ g/sample) or CD40-Ig (10 ⁇ g/sample) and resolved by PAGE and fluorography. Results presented are representative of 2 such experiments.
  • FIG. 6 A monoclonal antibody (mab), specific to the induced 39 Kd T h membrane protein, inhibited induction of B-cell RNA synthesis by PM Act . Resting B-cells and PM Act were cultured with 10 ⁇ g/ml each of anti- ⁇ / ⁇ , anti-CD3, CD40-Ig or MR1. RNA synthesis was determined as described in FIGS. 1A-1C. Results presented are the arithmetic means of triplicate cultures+/ ⁇ s.d., and are representative of 3 such experiments.
  • FIGS. 7A-7C Binding of CD40-Ig to human cell lines. A variety of human T-cell lines were exposed to biotin-labelled CD40-Ig, and binding was evaluated by flow cytometry.
  • FIGS. 8A-8B are identical to FIGS. 8A-8B.
  • FIG. 8 A Nucleotide sequence of CD40 cDNA from Stamenkovic et al., (1989) EMBO J. 8:1403-1410 (SEQ ID NOS. 1 & 2). The transmembrane region is underscored.
  • FIG. 8 B Schematic diagram of a plasmid that may be used to express CD40-Ig. The amino acid sequences at the site of fusion of ⁇ CD40 is shown below the diagrammed portion of CD40.
  • FIGS. 9A-9D Immunohistochemical localization and characterization of gp39+ cells in lymphoid organs.
  • FIG. 9A 4 days after secondary i.v. immunization with KLH; red stained cells are gp39 + cells localized in outer-PALS (o), around the terminal arteriole (t) and in the follicle (f) of the spleen [MR1+RG7-HRP; AEC].
  • FIG. 9B cryostat section of lymph node 6 days after secondary f.p. immunization with TNP-KLH; red stained cells are gp39 + cells localized in deep cortex and along the medullary cords.
  • FIG. 9C 3 days after KLH immunization; red membrane positive cells are CD4 + cells, while violet double staining cells are CD4 + expressing gp39 (arrow).
  • CD4 + cells are present in the follicle (f)
  • CD4 + cells expressing gp39 are found around terminal arterioles (t) and not in the primary follicle (f) [L3T4-HRP, AEC; MR1-AP, Fast blue]
  • FIG. 9C 3 days after KLH immunization; red membrane positive cells are CD4 + cells, while violet double staining cells are CD4 + expressing gp39 (arrow).
  • FIGS. 10A-10B Immunohistochemical visualization of T-B cell interaction in the spleen.
  • FIGS. 11A-11B Kinetics of gp39-positive T cells and KLH-AFC after primary and secondary immunization with KLH.
  • BCBA.F1 mice were injected i.v. with 100 ⁇ g KLH and sacrificed at the indicated time points.
  • Another group of BCBA.F1 mice was injected with 100 ⁇ g of KLH, boosted 16 weeks later with 100 ⁇ g KLH i.v. and sacrificed at the indicated time points.
  • Spleens were removed and immunohistochemistry and image analysis was performed as described in Materials and Methods section. Values represent mean ⁇ SD of number of positive cells per mm 2 from three mice. Closed triangles, KLH-AFC; closed circles, gp39-bearing cells.
  • FIG. 12 Kinetics of gp39 + cells and TNP-AFC are superimposable after immunization with TNP-Ficoll.
  • BCBA.F1 mice were injected i.v. with 20 ⁇ g TNP-Ficoll and sacrificed at the indicated time points. Spleens were removed and immunohistochemistry and image analysis was performed as described in the Materials and Methods section. Values represent mean ⁇ SD of number of positive cells per mm 2 from three mice. Closed squares, TNP-AFC; closed circles, gp39-bearing cells.
  • FIGS. 13A-13B Cytokine-PC and TNP-AFC develop according to similar kinetics after immunization with TNP-KLH.
  • BCBA.F1 mice were injected i.v. with 100 ⁇ g TNP-KLH and sacrificed at the indicated time points. Spleens were removed and immunohistochemical demonstration of IL-2, IL-4, IFN- ⁇ -PC and TNP-AFC was performed as described in Materials and Methods section. Values represent mean ⁇ SD of number of positive cells per mm 2 from three mice.
  • FIG. 14 Schematic representation of the activation and migration of T and B cells in the spleen during the TD immune response. Arrows indicate migration of B and T cells in the spleen.
  • FIG. 15 A Anti-gp39 inhibits the generation of primary anti-SRBC PFC.
  • mice were administered 200 ⁇ l of 1.0% SRBC i.v. on d0.
  • mice On d0, d2, and d4 mice were given either 100 or 500 ⁇ g of purified MR1 (hamster anti-murine gp39, purified from ascites by DEAE HPLC) or 500 ⁇ g of purified hamster Ig, i.p.
  • the control group consists of mice receiving the immunization, but no antibody treatment. Spleens were removed from the mice on d5 and the number of direct (IgM) anti-SRBC PFC was determined by a modification of the Jerne plaque assay. The data is representative of 3 such experiments.
  • FIG. 15 B Prolonged immune suppression of primary anti-SRBC responses is induced by the administration of anti-gp39.
  • mice were immunized with SRBC (200 ⁇ l of 1.0% SRBC, i.v.) and on d0, d2 and d4, received 250 ⁇ g of anti-gp39 ( ⁇ ) or 250 ⁇ g hamster Ig ( ⁇ ), i.p. The time of antibody administration is indicated by the black bar.
  • the anti-SRBC PFC response was determined on d5 post-immunization. Additional mice were challenged with antigen (0.2 ml of 1.0% SRBC i.v.) 7 d or 14 d after initial antigen immunization and anti-gp39 administration. The anti-SRBC PFC was then assayed 5 d later. The results are representative of three similar experiments.
  • FIGS. 16A-16B Anti-gp39 inhibits the generation of secondary anti-KLH antibody responses.
  • mice were immunized with KLH in CFA (50 ⁇ g/mouse i.v.) Three months after immunization, mice were given a soluble booster with 10 ⁇ g of KLH (i.v.). On d0, d2 and d4, immune mice received 250 ⁇ g of anti-gp39 i.p. (open bars) or 250 ⁇ g HIg (hashed bars). Serum from individual mice was collected on d7 (FIG. 16A) or d14 (FIG. 16B) post-antigenic challenge, pooled and levels of anti-KLH antibodies were determined using isotype specific ELISAs. Units represent arbitrary values based on the titration curve of a standard immune serum.
  • FIGS. 17A-17B Anti-gp39 inhibits the generation of primary and secondary antibody responses to heterologous immunoglobulins.
  • mice were immunized i.p. with 100 ⁇ g Chi-L6 absorbed on alum. On d0, d2 and d4, immune mice received 250 ⁇ g of anti-gp39 i.p. (open bars) or 250 ⁇ g HIg (hashed bars). Serum from individual mice was collected on d7 after initial immunization (for IgM; FIG. 17A) or antigenic challenge (for IgG 1 ; FIG. 17 B). The levels of anti-Chi-L6 IgM and IgG 1 antibodies were determined using antigen specific ELISAs. Units represent arbitrary values based on the titration curve or a standard immune serum. All experimental groups were titered from 1:100 to 1:100,000 and the titer ascertained based on multiple point analysis. The levels of anti-Chi-L6 antibodies in unchallenged controls were below detection. The results are representative of two separate experiments.
  • FIGS. 18A-18B Anti-gp39 administration does not inhibit the generation of primary antibody responses to TNP-Ficoll.
  • FIG. 18 A Mice (3/group) were immunized with 200 82 l 1% TNP-SRBC i.v. On d0, d2, and d4 mice received 250 ⁇ g anti-gp39 or HIg. On d6 mice were bled and the IgM anti-TNP antibody titers determined by TNP 16 -BSA ELISA.
  • FIG. 18 B Mice (3/group) were immunized with 25 ⁇ g TNP-Ficoll i.v. On d0, d2, and d4 mice received 250 ⁇ g anti-gp39 or HIg.
  • mice On d6 mice were bled and the IgM anti-TNP antibody titers determined by TNP 16 -BSA ELISA. Units represent arbitrary values based on the titration curve or a standard immune serum. All experimental groups were titered from 1:100 to 1:100,000 and the titer ascertained based on multiple point analysis. The anti-TNP titer of nonimmune mice was 390 units. The results are representative of two separate experiments.
  • FIG. 19 Anti-gp 39 administration does not functionally delete SRBC-specific T h .
  • mice were immunized with SRBC (200 ⁇ l of 1.0% SRBC i.v.) and administered anti-gp39 or HIg (on d0, d2, d4; 250 ⁇ g/d).
  • SRBC SRBC
  • anti-gp39 or HIg anti-gp39 or HIg
  • mice were removed and transferred (i.v., 50 ⁇ 10 6 /mouse) into irradiated recipients (600 rads) with/without 50 ⁇ 10 6 spleen cells from TNP-KLH primed (KLH/CFA 50 ⁇ g, i.v.) mice as a source of immune B cells.
  • mice were also immunized with TNP-SRBC (200 ⁇ l of 1.0% TNP/SRBC).
  • Serum IgG, anti-TNP titers were ascertained on d6 post-transfer using a TNP 2 -BSA ELISA. Units represent arbitrary values based on the titration curve of a standard immune serum. All experimental groups were titered from 1:100 to 1:100,000 and the titer ascertained based on multiple point analysis. The data are representative of two such experiments.
  • FIGS. 20A-20B In vivo clearance of hamster anti-gp39.
  • FIG. 20 A Serum (1.5 ⁇ g) was electrophoresed under non-reducing conditions, transferred to nitrocellulose and blotted with HRPO-conjugated RG7 (mouse anti-hamster ⁇ chain), followed by chemiluminescent detection. Areas of the blot corresponding to 150-165 kDa were scanned and digitized.
  • FIG. 20 B Titrations of serum were used to stain activated T h 1 to determine the amount of biologically active anti-gp39 present in the serum.
  • Activated T h 1 were stained with titrations of serum followed by FITC-anti-hamster ⁇ chain (RG7).
  • the percent anti-gp39 remaining in serum was deduced based on a standard curve of Mean Fluorescence Intensity (MFI) vs serum concentration, using d7 as 100%.
  • MFI Mean Fluorescence Intensity
  • FIGS. 21A-21B PM Act drive B cell cycle entry.
  • Murine resting B cells (3 ⁇ 10 4 /well) were cultured alone or with PM rest (2 ⁇ g/well), PM Act (2 ⁇ g/well), or anti-mIg (G ⁇ M IgG F(ab′) 2 ; 50 ⁇ g/ml) in a final volume of 50 ⁇ l for (FIG. 21A) 8 hours or (FIG. 21B) 24 hours at 37° C.
  • Wells were pulsed with 5 ⁇ Ci/well of 3 H-Udr for 2 hours, harvested and processed for liquid scintillation spectroscopy. Results are reported as cpm/culture, with standard error, and are an average of 5 such experiments.
  • FIG. 22 Measurement of cAMP i during T h dependent B cell activation.
  • Murine resting B cells (10 6 cells/sample) were cultured alone (denoted B) or with PM rest (10 ⁇ g/sample), PM Act (10 ⁇ g/sample), anti-MHC class II (M5; 50 ⁇ g/ml) or forskolin (50 ⁇ M) in a final volume of 100 ⁇ l for 15 minutes at 37° C. At the appropriate time, cells were analyzed for cAMP content by RIA as described in Methods and Materials. Results are reported as fmol cAMP/10 6 B cells, with standard error, and are an average of 3 such experiments.
  • FIG. 23 PM Act do not stimulate an increase in B cell Ca 2+ i .
  • Murine resting B cells (10 7 /ml) were loaded with Indo-1 (5 ⁇ M) for 30 minutes at 37° C. Cells were aliquoted at 10 6 /sample and maintained at 37° C. until FACS analysis. During analysis, PM rest (10 ⁇ g/sample), PM Act (10 ⁇ g/sample), anti-mIg (50 ⁇ g/ml) and ionomycin (5 ⁇ M) were added in a final volume of 100 ⁇ l and Indo-1 fluorescence was monitored over time. Results are reported as nM calcium, with standard error, and are an average of 3 such experiments.
  • FIG. 24 PM Act do not induce the phosphorylation of MARCKS in B cells.
  • Murine resting B cells (10 7 cells/ml) were labeled with 32 P-orthophosphate (0.5 ⁇ Ci/ml) for 1 hour at 37° C.
  • Cells were aliquoted (5 ⁇ 10 5 /sample) and then cultured alone or with PM Rest (10 ⁇ g/sample, PM Act (10 ⁇ g/sample or PMA (50 ⁇ g/ml) in a final volume of 100 ⁇ l for various time points at 37° C.
  • Cells were lysed and immunoprecipitated with an anti-MARCKS antiserum as described in Methods and Materials. Samples were analyzed through electrophoresis and autoradiography. The photograph shown is a representative example of 10 such experiments with the autoradiogram exposed for 18 hours at ⁇ 80° C.
  • FIG. 25 PM Act do not induce PKC translocation in B cells.
  • Murine resting B cells (5 ⁇ 10 5 /sample) were cultured with PM Rest (10 ⁇ g/sample, PM Act (10 ⁇ g/sample in a final volume of 100 ⁇ l for 30 minutes at 37° C.
  • PM Rest 10 ⁇ g/sample
  • PM Act 10 ⁇ g/sample in a final volume of 100 ⁇ l for 30 minutes at 37° C.
  • cells were cultured alone or with anti-mIg (G ⁇ M IgG F(ab′) 2 ; 50 ⁇ g/ml) or PMA (100 ng/ml) in a final volume of 100 ⁇ l for 5 minutes at 37° C.
  • Cells were then permeabilized, electrophoresed, and probed with an anti-PKC polyclonal antibody.
  • the photograph shown, including membrane fractions only, is a representative example of 8 such experiments and was exposed for 10 minutes.
  • FIGS. 26A-26B Induction of tyrosine phosphorylation during T h -dependent B cell activation.
  • FIG. 26 A Cells were then cultured in the presence and absence of anti-mIg (G ⁇ M IgG F(ab′) 2 ; 50 ⁇ g/ml) in a final volume of 100 ⁇ l for 15 minutes at 37° C. Cells were lysed, lysates were electrophoresed, and the blot was probed with an anti-phosphotyrosine monoclonal antibody. The photograph shown is a representative example of 10 such experiments and was exposed for 30 seconds.
  • FIG. 26 B The photograph shown is a representative example of 10 such experiments and was exposed for 30 seconds.
  • FIGS. 27A-27B Tyrosine phosphorylation is an active event.
  • Murine resting B cells (5 ⁇ 10 5 /sample) were preincubated for 30 minutes at 4° C. (Left panel) or 37° C. (Middle panel). Cells were then cultured with P rest (4 or 10 ⁇ g/sample) or PM Act (4 or 10 ⁇ g/sample) in a final volume of 100 ⁇ l for 1 hour at 4° C. or 37° C. Cells were lysed, electrophoresed, and probed with an anti-phosphotyrosine monoclonal antibody (FIG. 27 A). The photograph shown is a representative example of 12 such experiments and was exposed for 10 minutes.
  • Murine resting B cells were lysed, electrophoresed and probed with an anti-ERK polyclonal antiserum as described in Methods and Materials (Right panel FIG. 27 B). The photograph shown is a representative example of 12 such experiments and was exposed for 5 minutes.
  • FIG. 28 PM Act binding is not inhibited at 4° C. as measured by enhanced B cell cycle entry.
  • Murine resting B cells (5 ⁇ 10 5 /sample) were preincubated for 30 minutes at 4° C. or 37° C. Cells were then cultured with PM rest (10 ⁇ g/sample) or PM Act (10 ⁇ g/sample) in a final volume of 50 ⁇ l for 1 hour at 4° C. or 37° C. In addition, B cells were cultured alone or with anti-mIg (G ⁇ M IgG F(ab′) 2 ; 50 ⁇ g/ml) in a final volume of 50 ⁇ l for 15 minutes at 4° C. or 37° C. B Cells were then washed three times and RNA synthesis was measured. These results are reported as cpm/culture, with standard error, and are an average of 3 such experiments.
  • FIGS. 29A-29B Lack of detectable tyrosine phosphorylation in PM Act stimulated with PMA.
  • FIG. 30 Anti-gp39 inhibits PM Act induced tyrosine phosphorylation in B cells.
  • Murine resting B cells (5 ⁇ 10 5 /sample) were prewarmed for 30 minutes at 37° C. Cells were then cultured alone or with PM rest (10 ⁇ g/sample) or PM Act (10 ⁇ g/sample) which had been preincubated with anti-gp39, MR1, (50 ⁇ g/ml) or anti-TcR (50 ⁇ g/ml) in a final volume of 50 ⁇ l for 30 minutes at 4° C. Cultures were incubated in a final volume of 100 ⁇ l for 1 hour at 37° C. Cells were prepared for electrophoresis and probed with an anti-phosphotyrosine monoclonal antibody. The photograph shown is a representative example of 3 such experiments and was exposed for 10 minutes.
  • FIGS. 31A-31B Anti-CD40 induces a phosphotyrosine profile in human B cells.
  • Human B cells (5 ⁇ 10 5 /sample) were prewarmed for 30 minutes at 37° C. Cells were then cultured with anti-CD40 (1 ⁇ g/ml), mIgG (1 ⁇ g/ml or PMA (100 ng/ml) plus ionomycin (75 ng/ml) in a final volume of 100 ⁇ l for 5 minutes at 37° C. Cells were lysed, electrophoresed and probed with an anti-photyrosine monoclonal antibody (FIG. 31 A). The photograph shown is a representative example of 3 such experiments and was exposed for 30 minutes.
  • This anti-CD40/human B cell phosphotyrosine blot was stripped and reprobed with an anti-ERK polyclonal antiserum as described above (FIG. 31 B).
  • the photograph shown is a representative example of 3 such experiments and was exposed for 10 minutes.
  • FIG. 32 Effect of MR1 treatment on the primary immune response to Chi-L6.
  • FIG. 33 Effect of MR1 treatment on the secondary immune response to Chi-L6.
  • FIGS. 34A-34B Anti-gp39 treatment prevented the occurrence of CIA.
  • FIG. 34A Male DBA/1J mice 6 weeks of age were anesthetized and immunized with chick CII emulsified in a complete adjuvant prepared by combining Freund's incomplete adjuvant (Difco) and 2 mg/ml Mycobacterium tuberculosis (Ministry of Agriculture and Fisheries, Weybridge, Surrey, England). CII (200 ⁇ g) was injected in a single intradermal site at the base of the tail and then challenged with soluble CII (100 ⁇ g) 21 days later.
  • the mAb to gp39 antibody (MR1) is a hamster mAb to mouse gp39.
  • Antibody treatment (DEAE purified MR1 and HIg) was initiated 7 days after the primary immunization and maintained throughout the study (250 ⁇ g per mouse every 4 days). Mice (8 mice per group) were then routinely monitored for the development of clinical symptoms of CIA by inspection of distal joint inflammation.
  • MAI Mean Arthritis Index
  • FIG. 35 Anti-gp39 inhibits the secondary humoral immune response to CII.
  • Serum samples were analyzed for the presence of mouse CII-reactive IgG.
  • Each well of a polyvinyl microtiter plate was coated overnight at 4° C. with 100 ⁇ l of 5 ⁇ g/ml of chick CII and then blocked at incubation with PBS containing 1% FCS and 0.02% azide. After washing, a 100 ⁇ l aliquot of each serum, diluted in PBS, was added and the plates were incubated for 2 hours at 37° C., washed again, and incubated for a further 2 hours with 100 ⁇ l of alkaline phosphatase conjugated goat anti-mouse IgG 1 (Southern Biotechnology Inc., Birmingham Ala.).
  • FIG. 36 Time course indicating the induction of GVHD and the administration of anti-gp39.
  • mice induced with GVHD for 1 wk mice induced with mice induced with mice induced with GVHD for 2 wks
  • mice induced with GVHD for 1 wk while undergoing MR1 treatment mice induced with mice induced with 2 wk GVHD but only treated with the antibody for the first week of the disease.
  • FIG. 37 Indicator for splenomegaly in GVHD-induced mice.
  • MR1 anti-gp39 antibody
  • FIG. 38 A Hyperimmunoglobulin production in GVHD-induced mice and inhibition by anti-gp39.
  • Concentrations of IgA were obtained by culturing of spleen cells and performing ELISA assays on the supernatants.
  • FIG. 38 B Hyperimmunoglobulin production in GVHD-induced mice and inhibition by anti-gp39.
  • Concentrations of IgG1 were obtained by culturing of spleen cells and performing ELISA assays on the supernatants.
  • the present invention provides for substantially purified CD40CR; for soluble ligands of CD40CR, including antibodies as well as fusion molecules comprising CD40; and for methods of controlling B-cell activation.
  • the present invention provides for soluble ligands of CD40CR, including (i) fusion molecules comprising at least a portion of CD40 protein and (ii) antibodies or antibody fragments.
  • soluble indicates that the ligands of the invention are not permanently associated with a cell plasma membrane. Soluble ligands of the invention may, however, be affixed to a non-cellular solid support, including a lipid, protein, or carbohydrate molecule, a bead, a vesicle, a magnetic particle, a fiber, etc. or may be enclosed within an implant or vesicle.
  • CD40-Ig infra
  • MR1 infra
  • the ligands of the invention may be comprised in pharmaceutical compositions together with a suitable carrier.
  • the present invention provides for soluble fusion molecules that are ligands of CD40CR.
  • Such fusion molecules comprise at least a portion of CD40 protein attached to a second molecule.
  • the portion of CD40 preferably lacks the CD40 transmembrane domain.
  • a portion of CD40 protein which may be used according to the invention is defined as any portion which is able to bind to CD40CR, for example, such a portion may be shown to bind to the same protein as MR1 or CD40-Ig.
  • Second molecules which may be used include peptides and proteins, lipids, and carbohydrates, and, in preferred embodiments of the invention, may be an immunoglobulin molecule, or portion thereof (such as an Fv, Fab, F(ab′) 2 , or Fab′ fragment) or CD8, or another adhesion molecule, such as B7.
  • the second molecule may be derived from either a non-human or a human source, or may be chimeric.
  • the second molecule may also be an enzyme, toxin, growth factor, lymphokine, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound.
  • the fusion molecules of the invention may be produced by chemical synthesis or, preferably, by recombinant DNA techniques.
  • a nucleic acid sequence encoding at least a portion of CD40 protein may be combined with a nucleic acid sequence encoding a second molecule in a suitable expression vector, and then expressed in a prokaryotic or, preferably, eukaryotic expression system, such as a yeast, baculovirus, or mammalian expression system, including transgenic animals.
  • a prokaryotic or, preferably, eukaryotic expression system such as a yeast, baculovirus, or mammalian expression system, including transgenic animals.
  • CD40 protein may be expressed using recombinant DNA techniques and then may be chemically conjugated to a second molecule.
  • Fusion molecules comprising CD40 may be purified from preparative mixtures using electrophoretic techniques or affinity chromatography using ligand that binds to either CD40 or to the second molecule.
  • Ligands that bind to CD40 include, but are not limited to, anti-CD40 antibodies such as G28-5, as produced by the hybridoma having accession number HB9110 and deposited with the American Type Culture Collection, and CD40CR, described more fully in sections 5.2 and 5.3, infra. If the second molecule is an immunoglobulin or immunoglobulin fragment, an affinity column comprising anti-immunoglobulin antibody may be used; if the second molecule comprises an F c fragment, a protein A column may be used.
  • a portion of CD40 may be produced using a nucleic acid sequence that encodes a CD40 protein that is truncated upstream from the transmembrane domain.
  • a nucleic acid sequence may be prepared by digesting a plasmid containing a cDNA encoding CD40 antigen, such as that described in Stamenkovic et al., (1989), EMBO J. 8:1403-1410, with PstI (P) and Sau 3A (S3) restriction enzymes. The resulting P/S3 fragment may be subcloned into the same plasmid digested with P and Bam HI (B), to produce a truncated CD40 gene (see FIG. 8 B).
  • an expression vector used to produce ligands containing at least a portion of CD40 as well as immunoglobulin sequence may preferably comprise (i) a virally-derived origin of replication, a bacterial origin of replication, a bacterial selectable marker, and eukaryotic promoter and enhancer sequences separated from DNA sequences encoding an immunoglobulin constant region by restriction endonuclease sites which allow subcloning of DNA sequences encoding at least a portion of CD40, followed by a polyadenylation signal sequence (see FIG. 8 B.).
  • the truncated CD40 gene may be subcloned into an immunoglobulin fusion plasmid, such as that described in Aruffo et al., 1990, Cell 61:1303-1313, using an Mlu I and B digest, to form plasmid pCD40-Ig, which encodes the fusion molecule CD40-Ig (see FIG. 8 B).
  • CD40-Ig fusion protein may then be produced by transfecting the pCD40-Ig plasmid into COS cells to form a transient expression system.
  • CD40-Ig produced may be collected from the COS cell supernatant and purified by protein A column chromatography as described in Aruffo et al., 1990, Cell 161:1303-1313.
  • the soluble ligands of the invention may comprise antibody molecules, monoclonal antibody molecules, or fragments of these antibody molecules which contain an antigen combining site that binds to CD40CR.
  • Such ligands may further comprise a second molecule which may be a protein, lipid, carbohydrate, enzyme, toxin, growth factor, lymphokine, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound and may be linked to the antibody molecule or fragment.
  • the monoclonal antibody can be prepared against CD40CR using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
  • the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497) as well as other techniques which have more recently become available such as the human B-cell hybridoma technique (Kozbar et al., 1983, Immunology Today 4:72) and EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96) and the like are within the scope of the present invention.
  • Antibody fragments which contain the idiotype of the molecule could be generated by known techniques.
  • such fragments include but are not limited to: the F(ab′) 2 fragment which can be generated by treating the antibody molecule with pepsin; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′) 2 fragment; the F(ab′) 2 fragment which can be generated by treating the antibody molecule with papain; and the 2Fab or Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent to reduce the disulfide bridges.
  • the present invention also provides for chimeric antibodies produced by techniques known in the art, such as those set forth in Morrison et al., (1984) Proc. Natl. Acad. Sci. U.S.A. 81:6851-6855 or European Patent Application No. 85305604.2, publication No. 0173494 by Morrison et al., published Mar. 5, 1986.
  • Immunogen for the production of antibodies may be any source that contains CD40CR.
  • activated T h may be used as an immunogen.
  • substantially purified CD40CR prepared as set forth in section 5.3, infra, may be used. If activated T h are used as immunogen, antiserum may be tested for reactivity against activated but not resting T h cells.
  • the soluble ligand is the MR1 monoclonal antibody.
  • the following method was used to produce the MR1 monoclonal antibody, and may be used to generate other antibodies directed toward CD40CR.
  • the present invention also provides for ligands comprising monoclonal antibodies, and fragments thereof that are capable of competitively inhibiting the binding of MR1 to its target antigen or CD40-Ig to its receptor.
  • CD40CR may be characterized by (i) its ability to bind CD40, fusion molecules comprising at least a portion of CD40, and antibodies such as MR1; (ii) its functional characteristic of being able to stimulate B-cell cycle entry, proliferation, and differentiation; and (iii) its cellular distribution.
  • CD40CR may be characterized by its ability to bind to ligands such as CD40, fusion molecules comprising CD40, and antibodies directed toward CD40CR.
  • CD40-Ig and MR1 were shown to recognize the same 39 kD molecule. Both CD40-Ig and MR1 were found to immunoprecipitate a 39 kD protein from radiolabelled T h lysates (FIG. 5 B). Further, immunoprecipitation of the 39 kD protein with CD40-Ig removed the antigen recognized by MR1 from T h lysates.
  • CD40CR may also be characterized by its ability to stimulate B-cell cycle entry, proliferation, and differentiation.
  • the induction of B-cell activation may be measured by techniques such as [ 3 H]-uridine incorporation into RNA (as B-cells differentiate, RNA synthesis increases), or by [ 3 H]-thymidine incorporation, which measures DNA synthesis associated with cell proliferation.
  • interleukin-4 IL-4
  • IL-4 may be added to the culture medium at a concentration of about 10 ng/ml.
  • B-cell activation may be measured as a function of immunoglobulin secretion.
  • CD40CR in substantially purified form, or as present in PM, or otherwise, may be added to resting B-cells together with IL-4 (10 ng/ml) and IL-5 (5 ng/ml). After three days of culture, an additional volume of culture medium may be added. On day 6 of culture, supernatant (SN) from individual cultures may be harvested and quantitated for IgM and IG 1 as described in Noelle et al., (1991) J. Immunol. 146:1118-1124.
  • CD40CR may also be characterized by its cellular distribution. For example, CD40-Ig was observed to bind to activated, but not resting T h 1, as assessed by flow cytometry (FIGS. 3 A- 3 B). Furthermore, CD40-Ig was observed to bind to Jurkat cells, HSB2 cells, and activated T-cells from human peripheral blood, but did not appear to bind significantly to CEM cells, HPBALL cells, or murine thymoma cells.
  • test cells may be evaluated by flow cytometry as follows. Test cells may be tested in parallel with resting (negative control) and activated (positive control) T h cells. All cells may be incubated at a concentration of about 1 ⁇ 10 5 cells/50 ⁇ l with ligand (e.g. CD40-Ig or MR1) for 20 minutes at 4° C., followed by FITC-conjugated anti-ligand antibody. Propidium iodide may be added to all samples to a final concentration of 2 ⁇ g/ml. Flow cytometric analysis may then be performed, for example on a BD FACSCAN. After positive gating of cells by forward versus side scatter, and by red negativity (for propidium iodide exclusion), and the log green fluorescence of viable cells may be ascertained.
  • ligand e.g. CD40-Ig or MR1
  • FITC-conjugated anti-ligand antibody e.g. CD40-Ig or MR1
  • the present invention provides for substantially purified CD40CR.
  • CD40CR may be prepared from cells bearing CD40CR, such as activated helper T-cells, Jurkat,and HSB2 cells, by the following method.
  • Plasma membranes may be prepared from appropriate cells, such as activated T h 1 cells, by discontinuous sucrose gradient sedimentation, as described in Noelle et al., 1991, J. Immunol. 146:1118-1124.
  • CD40CR may then be isolated by dissociating the crude membrane extract with mild detergent, and then performing size exclusion chromatography followed by either affinity chromatography using appropriate ligands (e.g. MR1 or CD40-Ig) bound to a solid support, immunoprecipitation (e.g. by CD40-Ig or MR1), and/or gel electrophoresis.
  • the resulting protein may be expected to have a molecular weight of about 39 kD.
  • the present invention provides for a soluble CD40CR (i.e. cell-free) which may be comprised in pharmaceutical compositions together with a suitable carrier. It further provides for CD40CR which is linked to a second molecule which may be a peptide, protein, lipid, carbohydrate, enzyme, toxin, growth factor, lymphokine, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound.
  • a soluble CD40CR i.e. cell-free
  • CD40CR which is linked to a second molecule which may be a peptide, protein, lipid, carbohydrate, enzyme, toxin, growth factor, lymphokine, antiproliferative agent, alkylating agent, antimetabolite, antibiotic, vinca alkaloid, platinum coordinated complex, radioisotope, or a fluorescent compound.
  • the present invention further provides for substantially purified CD40CR which has been prepared by chemical synthesis or recombinant DNA techniques.
  • the gene for CD40CR may be isolated by inserting cDNA prepared from activated helper T-cells into the ⁇ gt10 expression system, and then screening with MR1 or CD40-Ig binding to identify CD40CR-expressing clones.
  • cDNA prepared from activated helper T-cells may be transfected into COS cells, the supernatants of which may be screened with MR1 or CD40-Ig to identify CD40CR producers.
  • the gene for CD40CR may be then used to express CD40CR using expression systems known in the art.
  • the present invention provides for methods of controlling B-cell activation that utilize ligands that bind to CD40CR.
  • a method of inhibiting B-cell activation comprising exposing a mixture of B-cells and T h cells to an effective concentration of ligand that binds to CD40CR.
  • Ligands that may be used are described supra in section 5.1.
  • the method of the invention may be practiced in vitro or in vivo.
  • An effective concentration refers to a concentration of a ligand that inhibits B-cell activation, measured by any technique known in the art (including those set forth in section 5.2, supra) by at least about 30 percent, and preferably by about 75 percent.
  • CD40-Ig may be used as ligand, in which case an effective concentration may be at least about 10 ⁇ g/ml.
  • the monoclonal antibody MR1 may be used, in which case an effective concentration may be at least about 10 ⁇ g/ml.
  • an effective concentration of ligand may refer to plasma concentration of ligand or to a local concentration. For example, it may be desirable to inhibit B-cell activation in a localized area in order to limit the effects on the immune system as a whole.
  • the invention provides for a method of treating a subject suffering from a disorder associated with B-cell activation, comprising administering to the subject a therapeutic amount of ligand that binds to CD40CR.
  • a subject may be a non-human or, preferably, a human animal.
  • Disorders associated with B-cell activation include, but are not limited to, allergy (including anaphylaxis); autoimmune conditions (see Section 12, infra, which demonstrates the effectiveness of CD40CR ligand in a murine model of rheumatoid arthritis) including drug induced lupus, systemic lupus erythematosus, adult rheumatoid arthritis, juvenile rheumatoid arthritis, scleroderma, Sjogren's Syndrome, etc.; and viral diseases that involve B-cells, including Epstein-Barr infection, and retroviral infection including infection with a human immunodeficiency virus.
  • allergy including anaphylaxis
  • autoimmune conditions see Section 12, infra, which demonstrates the effectiveness of CD40CR ligand in a murine model of rheumatoid arthritis
  • drug induced lupus including drug induced lupus, systemic lupus erythematosus, adult rheum
  • B-cell activation is associated with the induction of human immunodeficiency virus replication from latency, it may be desirable to administer the ligands of the invention to HIV positive individuals who have not yet developed AIDS or ARC.
  • the present invention further provides for methods comprising administering an effective amount of CD40CR ligand to reduce a primary and/or secondary humoral immune response to thymus dependent antigen, which methods do not substantially alter the humoral response to thymus-independent type II antigens, (see Section 9, infra).
  • the CD40CR ligand may be a monoclonal antibody administered to a human subject at a dose between 0.1 mg and 20 mg/kg body weight.
  • CD40CR ligand has been demonstrated to prevent primary and secondary humoral responses to heterologous antibody preparations, thereby improving the therapeutic potential of such heterologous antibody preparations in patient treatment by permitting repeated administration of heterologous antibody without invoking substantial humoral immunity.
  • CD40CR ligand may be used to prevent or ameliorate graft versus host disease in a subject in need of such treatment. Accordingly, the invention provides for methods of inhibiting graft versus host disease in a subject in need of such treatment comprising an effective amount of CD40CR ligand to a subject in need of such treatment. See Sections 11 and 13 for data demonstrating that anti-gp39 antibody was able to inhibit grafted T cells from inducing host B cell activation.
  • Ligands may be administered, in a suitable pharmaceutical carrier, by any method known in the art, including intravenous, intraperitoneal, subcutaneous, intrathecal, intraarticular or intramuscular injection, and oral, intranasal, intraocular and rectal administration, and may be comprised in microspheres, liposomes, and/or sustained release implants.
  • a therapeutic amount of ligand is defined as an amount which significantly diminishes the deleterious clinical effects of B-cell activation, and may vary among ligands used and conditions treated. If CD40-Ig is used, therapeutic concentration may be about 10 ⁇ g/ml either systemically (plasma concentration) or locally. If MR1 is used, a therapeutic concentration may be about 10 ⁇ g/ml either systemically (plasma concentration) or locally.
  • the above methods may utilize a ligand comprising a toxin or antimetabolite such that T h cells are killed or damaged and B-cell activation is decreased as a result of T h cell destruction.
  • the ligands of the invention may also be used to label activated T cells, a technique which may be useful in the diagnosis of T cell disorders.
  • ligand comprising an enzyme, radioisotope, fluorescent compound or other detectable label may be exposed to T cells in vitro or in vivo and the amount of binding may be quantitated.
  • the ligands of the invention may also be used to deliver substances, e.g. growth factors, to activated T-cells.
  • the present invention provides for methods of controlling B-cell activation that utilize CD40CR or a molecule comprising CD40CR, prepared as described in section 5.3, supra.
  • a method of promoting B-cell activation comprising exposing B-cells to an effective concentration of CD40CR.
  • the method may be practiced in vivo or in vitro.
  • An effective concentration refers to a concentration of receptor that induces B-cell activation, measured by any technique known in the art (including those set forth in section 5.3, supra) by at least about 30 percent.
  • the concentration of CD40CR may be about 10 ⁇ g/ml locally or systemically.
  • the invention provides for a method of treating a subject suffering from an immunodeficiency disorder associated with diminished humoral immunity, comprising administering to the subject a therapeutic amount of CD40CR.
  • a subject may be a non-human or, preferably, a human animal.
  • Immunodeficiency disorders associated with diminished humoral immunity include acquired immunodeficiency syndrome, immunodeficiency associated with malignancy or cachexia, iatrogenic immunodeficiency caused, for example, by chemotherapy or radiation therapy, as well as genetic disorders involving humoral immunity.
  • CD40CR may be administered,in a suitable pharmaceutical carrier, by any method known in the art, including intravenous, intraperitoneal, subcutaneous, intrathecal, intraarticular, or intramuscular injection, and oral,intranasal, intraocular, and rectal administration and may be comprised in microspheres, liposomes, and/or sustained release implants.
  • a therapeutic amount of CD40CR for CD40 is defined as that amount which increases immunoglobulin production by at least about 30 percent.
  • CD40CR may be conjugated to a toxin, and then administered to a subject under circumstances in which it would be preferable to destroy B-cells that express CD40. Examples of such circumstances include patients receiving organ transplants or suffering from multiple myeloma or another B-cell malignancy,or from autoimmune disease.
  • CD40CR may also be used to label B-cells expressing CD40, a technique which may be useful in the diagnosis of B-cell disorders.
  • receptor linked to an enzyme, radioisotope, fluorescent compound or other detectable label may be exposed to B-cells in vivo or in vitro and the amount of binding may be quantitated.
  • CD40CR may also be used to deliver molecules that are linked to it to B-cells.
  • CD40CR A Novel Receptor, CD40CR, on Activated Helper T-Cells Binds CD40 and Transduces the Signal for Cognate Activation of B-Cells
  • mice Female DBA/2J mice (Jackson Laboratories, Bar Harbor, Me.) were used for the preparation of filler cells to support the growth of T h clones and in the preparation of resting B-cells.
  • T h 1 a I-A d -restricted, rabbit Ig-specific T h 1 clone (Kurt-Jones et al., (1987) J Exp Med 166:1774-1787) was obtained from Dr. David Parker, University of Mass. at Worcester. D1.6 will be referred to herein as T h 1.
  • T h 1 were cultured (8 ⁇ 10 6 /well) in cluster wells (6 well, Corning, N.Y.) coated with 40 ⁇ g/4 ml of PBS/well with anti-CD3 for 16 hours, as described in (Noelle et al., (1991) J. Immunol. 146:1118-1124).
  • Plasma membranes were prepared by discontinuous sucrose gradient sedimentation, as described in (Noelle et al., (1991) J. Immunol. 146:1118-1124).
  • Resting splenic B-cells were prepared by sedimentation on discontinuous Percoll gradients, as described in (Defranco et al., (1982) J. Exp. Med. 155:1523). Cells isolated from the 70-75% (density of 1.087-1.097) Percoll interface were typically >95% mIg + , had a uniform, low degree of near forward light scatter and were unresponsive to Con A.
  • anti-CD3:145-2C11 (Leo et al., (1987) Proc. Natl. Acad. Sci. USA 84:1374-1378); anti- ⁇ , ⁇ :H57-597; anti-CD4: GK1.5 (Wilde et al., (1983) J. Immunol. 131:2178-2183); anti-ICAM:YN1/1.7.4 (Prieto et al., (1989) Eur. J. Immunol.
  • the CD40 fusion protein was prepared by digesting a plasmid containing a cDNA encoding the CD40 antigen (Stamenkovic and Seed, (1989) EMBO J. 8:1403-1410) with the restriction enzyme Pst I (P) and Sau 3A (S3). This P/S3 fragment was subcloned into the same plasmid digested with P and Bam H1 (B). This allowed the preparation of the CD40 ⁇ which encoded a CD40 protein truncated upstream from the transmembrane domain. The DNA fragment encoding a CD40 ⁇ was then subcloned into the immunoglobulin fusion plasmid (Aruffo et al. (1990), Cell.
  • the CD40-Ig fusion protein was produced by transient transfection in COS cells and purified on a protein A column as described in (Aruffo et al., (1990) Cell. 61:1303-1313).
  • Interleukin 4 Recombinant mouse IL4 was generously provided by Drs. C. Maliszewski and K. Grabstein, Immunex Corporation, Seattle, Wash.
  • Interleukin 5 Recombinant mouse IL5 was purchased from R&D Research, Sarrento, Calif.
  • 3 ⁇ 10 4 resting B-cells were cultured in 50 ⁇ l of cRPMI in A/2 microtiter wells (Costar, Cambridge, Mass.). To these wells, 0.5 ⁇ g of T h 1 or T h 2 membrane protein was added. From 42-48 hrs, wells were pulsed with 2.5 ⁇ Ci of 3 H-uridine (New England Nuclear, Boston Mass.), harvested, and the radioactivity determined by liquid scintillation spectroscopy. The results were expressed as cpm/culture +/ ⁇ s.d.
  • Resting B-cells were cultured as described above. To culture wells, 0.5 ⁇ g of T h 1 membrane protein, IL4 (10 ng/ml) and IL5 (5 ng/ml) were added. On day three of culture, an additional 50 ⁇ l of cRPMI was added. On day six of culture, SN from individual wells were harvested and quantitated for IgM and IgG 1 , as described in (Noelle et al., (1991) J. Immunol. 146:1118-1124).
  • T h Resting and activated T h (16 hours with anti-CD3) were harvested and incubated at 1 ⁇ 10 5 cells/50 ⁇ l with fusion protein for 20 minutes at 4° C., followed by FITC-conjugated goat anti-human (h)IgG (25 ⁇ g/ml; Southern Biotechnology, Birmingham, Ala.). To all samples, propidium iodide was added at final concentration of 2 ⁇ g/ml. Flow cytofluorometric analysis was performed on a BD FACSCAN. After positive gating of cells by forward versus side scatter, and by red negativity (for propidium iodide exclusion), the log green fluorescence of viable cells is was ascertained. At least 5,000 viable cells were analyzed for the determination of percent positive cells and MFI. Staining with MR1 employed FITC-conjugated RG7, a mouse anti-rat/hamster ⁇ chain mab.
  • T h 1 were rested or activated with insolubilized anti-CD3 for 16 hrs.
  • Proteins from resting and activated T h (20 ⁇ 10 6 /ml) were labelled with 1 mCi of [ 35 S]-methionine/cysteine for one hour, at which time they were washed twice in RPMI/10% FCS and the cell pellet was lysed in extraction buffer, as described (Noelle et al., (1986) J. Immunol. 137:1718-1726).
  • Purified antibodies or fusion proteins (1-10 ⁇ g) were added to 500 ⁇ l of lysate (5 ⁇ 10 6 cell equivalents) at 4° C. for 16 hours.
  • the lysates were transferred to tubes containing 50 ⁇ l of packed Protein A-sepharose.
  • the pelleted Protein A-Sepharose was resuspended and tubes were incubated at 4° C. for 1 hr with agitation. The samples were then washed 3 ⁇ with high stringency wash buffer.
  • the pelleted protein A-Sepharose was resuspended in 30 ⁇ l of SDS sample buffer and run on a 10% polyacrylamide gel. After running the gel, the gel was fixed and fluorography performed.
  • CD40-Ig The addition of CD40-Ig to culture caused a dose-dependent inhibition of B-cell RNA synthesis that was induced by PM Act from T h 1 and T h 2 (FIG. 1 B).
  • a CD7E-Ig fusion protein (Damle and Aruffo, (1991) Proc. Natl. Acad. Sci. USA 88:6403-6407) was without effect even when used at 25 ⁇ g/ml.
  • CD40-Ig inhibited the activation of B-cells by T-independent activators
  • B-cells were cultured in the presence of LPS and CD40-Ig.
  • RNA synthesis was assessed (FIG. 1 C).
  • CD40-Ig was ineffective at inhibiting B-cell activation by LPS, yet inhibited the response of B-cells to PM Act .
  • CD40-Ig was added at the initiation of culture, or on subsequent days of culture.
  • T h 1 were activated for 16 hours with insolubilized anti-CD3, harvested and irradiated. The irradiated T h 1 were cultured with B-cells in the presence of IL4 and B-cell proliferation was determined on day 3 of culture. An exogenous source of IL4 was required to achieve B-cell proliferation with T h 1, because T h 1 do not produce IL4 (Noelle et al., (1989) J. Immunol. 143:1807-1814).
  • CD40-Ig inhibited the induction of B-cell proliferation by irradiated T h in a dose-dependent manner, similar to that observed with PM Act (FIG. 2 B).
  • CD40-Ig Detected a Molecule Expressed on Activated, but not Resting T h
  • T h 1 express is a binding protein for CD40
  • resting and activated (16 hours) T h 1 were stained with CD40-Ig or CD7E-Ig, followed by FITC-anti-HIgG. Binding of CD40-Ig was assessed by flow cytometry (FIGS. 3 A- 3 B). T h 1 that were activated for 16 hours with anti-CD3, but not resting T h 1, stained 56% positive with CD40-Ig, but not with the control CD7E-Ig.
  • T h 1 proteins were biosynthetically labelled with [ 35 S]-methionine/cysteine and proteins immunoprecipitated with CD40-Ig or CD7E-Ig.
  • the immunoprecipitated proteins were resolved by SDS-PAGE and fluorography (FIG. 4 ).
  • anti-class I mab immunoprecipitated bands at 55 kD and a low molecular weight band, ⁇ 2 microglobulin. In the absence of mab, no prominent bands were visible.
  • a 39 kd band was also immunoprecipitated from activated T h that were vectorially labelled with 125 I, confirming that the 39 kD protein was a membrane protein.
  • Mabs specific to antigens selectively expressed on activated versus resting T h were developed to identify T h molecule(s) responsible for the T h effector phase activity.
  • One such mab, MR1 recognized an antigen that was selectively expressed on activated T h 1.
  • flow cytometry and blocking studies were performed.
  • CD40-Ig and MR1 stained approximately 56% and 61%, respectively, of activated, but not resting Th FIG. 5 A.
  • MR1 but not another hamster anti-T cell mab, anti- ⁇ / ⁇ TCR, blocked the staining of activated T h 1 with CD40-Ig, in a dose-dependent manner.
  • CD40-Ig and MR1 recognized overlapping or identical epitopes on the 39 kD Th protein.
  • the antigen that bound MR1 was identified by immunoprecipitation of proteins from radiolabelled Th lysates. Both CD40-Ig and MR1 immunoprecipitated a 39 kD protein (FIG. 5 B).
  • immunoprecipitation of the 39 kD protein with CD40-Ig removed the antigen recognized by MR1 from radiolabelled lysates of activated T h supporting the tenet that the MR1 antigen and the CD40 binding protein were identical.
  • T h surface molecules LFA-1, CD4, ICAM-1, CD3, ⁇ , ⁇ TCR
  • CD40-Ig or a maB specific to the CD40 binding protein blocked T h -dependent B-cell activation in a dose-dependent manner.
  • the CD40 binding protein was identified as a 39 kD protein that is selectively expressed on the membranes of activated, but not resting T h . Both CD40-Ig and a mab specific to the 39 kD CD40 binding protein blocked B-cell activation by PM Act .
  • the ligand for CD40 is a 39 Kd protein that is expressed on activated, but not resting T h .
  • Biochemical studies indicate that the 39 kD protein is a single chain molecule since electrophoretic migration was not influenced by reducing agents.
  • both activated T h 1 and T h 2 express the 39 kD CD40 binding protein. This is consistent with the functional studies that show both T h 1 and T h 2 induce B-cell cycle entry.
  • cDNA encoding CD proteins in the MW range of 39 kD (CD 53, CD27 and CD69) were transiently transfected into COS cells and the cells were tested for CD40-Ig binding. None of the transfected COS cells expressed proteins that bound CD40-Ig. It is therefore suspected that the 39 kD protein is not one of these CD proteins.
  • CD40 is a member of the nerve growth factor receptor (NGFR) family by virtue of the presence of four cysteine-rich motifs in its extracellular region. Signaling through CD40 by mab has been shown (Uckun et al., (1991) J. Biol. Chem. 266:17478-17485) to involve the activation of tyrosine kinases resulting in the increased production of inositol trisphosphate and the activation of at least four distinct serine/threonine kinases. Based on information obtained from signaling through other members of the NGF receptor family, it is anticipated that interaction between activated T h and B will result in many of the same biochemical processes.
  • NGFR nerve growth factor receptor
  • CD40 Ig fusion protein was conjugated with biotin using biotin-succinimide (Sigma). Flow cytometry analysis was then performed by tow-step staining using phycoerythrin (PE)-strepavidin (Bectin-Dickinson) with a Coulter Epics C instrument. Representative results of screening multiple T cell lines is presented below. The Jurkat and HSB2 cell lines were found to bind specifically, whereas other T cell lines including CEM, HPBALL, and murine thymoma did not bind the CD40 Ig fusion protein (FIGS. 7 A- 7 C).
  • T h cells Upon activation, T h cells express the ligand for CD40, gp39, which is essential for T h cell dependent B cell activation.
  • the cytokines produced by activated T h cells have a regulatory role in B cell differentiation.
  • immunohistochemical techniques were used to investigate the in vivo time course and localization of gp39-expression and cytokine-production in relation to specific antibody production.
  • mice were bred at the TNO breeding facilities, Rijswijk, The Netherlands. Animals were used at 16-24 weeks of age and were kept under a standard protocol with free access to pelleted food and acidified water (pH 3). Experiments were performed under auspices of the Dutch Veterinary Inspection, as described in the law on animal experiments.
  • Alkaline phosphatase (AP; P-6774, type VII-T, 1020 U/mg protein), 3-amino-9-ethylcarbazole (AEC; A-5754), Complete Freunds Adjuvant (CFA), 3,3-diaminobenzidine-tetrahydrochloride (DAB), Fast blue BB Base (F-0125), Fast red, horseradish peroxidase (HRP), Incomplete Freunds Adjuvant (IFA), levamisole, naphthol AS-MX phosphate (3-hydroxy-2-naphtoic acid 2,4-dimethyl-anilide), were obtained from Sigma, St. Louis, Mo., USA.
  • N-hydroxysuccinimidyl-(biotinamido)-hexanoate and MHS maleimidohexanoyl-n-hydroxysuccinimide ester
  • ⁇ -galactosidase ⁇ -gal; E. coli -derived ⁇ -D-galactoside galactohydrolase, MW 540 KD
  • X-Gal 5-bromo-4-chloro-3-indolyl- ⁇ -D-galactopyranoside
  • TNP-Ficoll and TNP-KLH were prepared as previously described (Claassen et al. (1986) Eur. J. Immunol. 16:271), Claassen et al. (1986) Eur. J. Immunol. 16:492).
  • the rat mAb Lyt2+ (CD8) (clone 53.6.7.2) (Ledbetter et al. (1979) Immunol. Rev. 47:63) and L3T4 (CD4)(clone GK-1.5) (Dialynas et al. (1983) J. Immunol. 131:2445) were used as cell markers.
  • the control hamster antibodies and ascites from the cell lines MR1 Noelle et al.
  • the cells of the rat mAb 11B11, directed to IL-4, and IL-4 were a kind gift of Dr. W. E. Paul, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, N.I.H., Bethesda, Md. (Ohara et al. (1985) Nature 315:333).
  • the IL-2 specific mAb, S4B6, was a kind gift of Dr. T. Mosmann (Mosmann (1986) J. Immunol. 136:2348).
  • Control rat mAb (2A4, 1G11) were a kind gift of Dr. A.
  • mice were injected i.v. with 100 ⁇ g of KLH or TNP-KLH or 20 ⁇ g of TNP-Ficoll and killed after 0, 1, 2, 3, 4, 5, 6, and 7 days.
  • Another group of mice was injected with 100 ⁇ g of KLH, boosted 16 weeks later with 100 ⁇ g TNP-KLH and killed after 0, 1, 2, 3, 4, 5 and 7 days.
  • mice were immunized I.p. with TNP-KLH in CFA, boosted 4 weeks later I.p. with 20 ⁇ g TNP-KLH in IFA and killed 6 days after injection. Spleens and popliteal lymph nodes were removed and immediately frozen in liquid nitrogen and stored at ⁇ 70° C.
  • Splenic cryostat sections ( ⁇ 20° C., 8 ⁇ m), one for every mouse, were picked up on the same glass slide and kept overnight under high humidity at RT. Slides were air-dried and stored in air-tight boxes until use. Slides were fixed for 10 minutes in acetone containing 0.02% H 2 O 2 . Slides were incubated horizontally overnight at 4° C. with primary cytokine-specific antibody-conjugates diluted in PBS containing 0.1% BSA and titrated to obtain optimal results.
  • Immunohistochemical demonstration of gp39 was performed in two ways: first, with a gp39-specific hamster mAb, MR-1, followed by a hamster-Ig-specific mAb, RG-7, conjugated to peroxidase; second, with a fusion-protein of the gp39 receptor, CD40-IgG1 (Noelle et al. (1992) Proc. Natl. Acad. Sci. USA 89:6550), followed by a human-IgG1-specific mAb conjugated to peroxidase.
  • spleen sections were incubated simultaneously with MR-1-AP and L3T4-HRP or Lyt2 + -HRP. Detection of KLH-specific antibody forming cells (KLH-AFC) and TNP-AFC were detected according to previously described methods (Claassen et al. (1986) Eur. J. Immunol. 16:271), (Claassen et al. (1986) Eur. J. Immunol. 16:492). Slides were washed with PBS (three times 5 minutes) and immunohistochemical revelation was performed as described previously; AP (Claassen et al. (1988) J. Histochem.
  • Antibody-forming cells, gp39 + cells and cytokine-PC were counted, and image-analysis was performed as previously described (Van den Eertwegh (1991) J. Immunol. 147:439). A minimum of three sections of each mouse were examined.
  • MR1 blocked the staining with CD40-IgG1 in a dose-dependent manner, confirming the fact that MR1 and CD40-IgG1 recognize the same molecule, gp39 (Noelle et al. (1992) Proc. Natl. Acad. Sci. USA 89:6550).
  • c Cryostat sections were prepared from spleens and immunohistochemical demonstration of gp39 was performed with an anti-gp39 mAb, or with CD40-Ig followed by secondary peroxidase-conjugates.
  • Cells were counted in each anatomical compartment d of the spleen: Follicular corona, Fc; germinal center, GC; inner-PALS, iP; outer-PALS, oP; marginal zone, MZ; terminal arterioles, TA.
  • e Values represent mean ⁇ SD number of positive cells in each compartment of spleen sections from three mice.
  • Gp39 + Th cells were found predominantly in the outer-periarteriolar lymphocyte sheaths (PALS) and around the terminal arterioles (TA) of the spleen (Table 1, FIG. 9A; FIG. 10 B).
  • Double staining for CD4 + and gp39 clearly revealed that CD4 + cells in primary follicles were not gp39 positive (FIG. 9 C). After secondary immunization, only a few gp39 + cells were observed in the follicular corona, but not in the GC of secondary follicles (FIG. 9 A).
  • lymph node sections of TNP-KLH immunized mice revealed that gp39 + cells were localized in the deep cortex and along the medulary cords (FIG. 9 B). Incidently, gp39 + cells were observed in the corona, but not in the GC, of follicles in lymph nodes.
  • Th cell derived cytokines have a decisive role in isotype-selection (Snapper et al. (1987) Science 236:944), Snapper et al. (1988) Immunol. Rev. 102:51; Stevens et al. (1986) Nature 334:255).
  • Th1/Th2 Th subset
  • the IL-4-specific mAb (11B11) conjugated to ⁇ -gal was used for the in situ demonstration of IL-4-PC. After immunohistochemical revelation, IL-4-PC characterized by a turquoise cytoplasm were detected (FIG. 9 d ). No staining was observed in control slides from any specimen treated by omission or substitution of the primary antibody. Preincubation of 11B11 conjugated to ⁇ -gal with recombinant IL-4 inhibited the staining of IL-4-PC, in a dose-dependent manner. Moreover, the specificity of the staining was confirmed on cytospin preparations of cells from an IL-4-producing cell line (X6310-IL-4).
  • IL-2-PC were demonstrated with an IL-2-specific mAb (S4B6) conjugated to HRP and were characterized by a red stained cytoplasm, respectively.
  • Control immuno-conjugates showed no staining.
  • the specific staining was inhibited by recombinant IL-2 in a dose dependent manner.
  • specificity was confirmed on cytospin preparations of cells from an IL-2-producing cell line (X6310-IL-2).
  • Gp39 + cells are a prerequisite for B cell activation in vitro (Noelle et al. (1992) Proc. Natl. Acad. Sci. USA 89:6550) and TD antibody responses in vivo. Thereafter, cytokine-producing Th cells are thought to be active as regulators of antibody responses.
  • gp39 + cells have the capacity to produce cytokines, as was suggested by in vitro experiments (Hodgkin et al. (1991) J. Immunol. 147:3696), we performed double-staining experiments.
  • MR-1 (gp39) was conjugated to AP, whereas S4B6 (IL-2) and 11B11 (IL-4) were conjugated to HRP and ⁇ -galactosidase, respectively.
  • Double staining for IL-2 and gp39 revealed red stained cells producing IL-2, blue stained gp39 + cells and violet double staining cells, representing gp39 + cells producing IL-2.
  • gp39 + Th cells have been found to be essential for the activation of B cells in vitro and in vivo, their anatomical localization in relation to resting and antibody-producing B cells was examined. Double immunohistochemical staining for resting B cells (membrane IgM-bearing) or B plasma blasts (cytoplasmic Ig) and gp39, revealed that the majority of gp39 + Th cells were co-distributed amongst both B cell types in the outer PALS and TA. In addition, when antibody-forming B cells, specific for the immunizing antigen, were revealed, the majority of the KLH-AFC/TNP-AFC were found in intimate contact (juxtaposition) with the gp39 + Th cells (FIG. 10 A). Also in the immune response against TNP-Ficoll we observed antigen-specific B cells (TNP-AFC) co-localizing with gp39 + cells in the outer-PALS and around the TA (FIG. 10 B).
  • the present study demonstrates that during in vivo antibody responses gp39 expression and cytokine-production develop simultaneously and are predominantly localized in the outer-PALS and around the TA of the spleen.
  • the gp39 + cells and cytokine-PC are found in juxtaposition to antigen-specific B cells.
  • IL-4 has been suggested to play a role to B cell switching to IgG1 and in the propagation of IgG1 + AFC (Stevens et al. (1986) Nature 334:255). Consequently, the observed juxtaposition of IL-4-PC to KLH-specific B cells may represent Th2-B cell interactions which stimulate the preferential development of IgG1 + cells.
  • incidentally ⁇ 1%
  • gp39 + cells in the corona of the follicles, but no gp39 + cells or cytokine-producing T cells were found in the GC.
  • FIG. 14 shows a model for the development of TD antibody responses in the spleen based on presented data and on the localization and migration of immune cells (Van den Eertwegh et al. (1992) Crit. Rev. Immunol. 11:337).
  • TD antigens are presented by interdigitating cells in the PALS, leading to increasing numbers of antigen-specific T cells which subsequently encounter antigen-specific B cells in the PALS, forming T-B cell conjugates.
  • CD4 + Th cells will be activated by antigen-presenting B cells and express CD40CR. Gp39 will trigger B cell growth and differentiation.
  • activated B cells migrate to the follicles to undergo follicular processes, such as B cell selection, somatic mutation, affinity maturation and memory formation (Liu et al. (1992) Immunol. Today 13:17; Kosco et al. (1992) Immunol. Rev. 126:63).
  • Other activated B cells migrate to the TA and differentiate into antigen-specific AFC regulated by activated cytokine-producing T cells. After secondary immunization, antigen-specific memory B cells acquire the antigen in the follicle, where it is presented by follicular dendritic cells in the form of immune complexes (Liu et al. (1992) Immunol. Today 13:17; Kosco et al.
  • the relatively low frequencies of gp39 + T cells could be due to the shorter lasting expression of gp39 by T cells (Hodgkin et al. (1990) J. Immunol. 145:2025; Lederman et al. (1992) J. Exp. Med. 175:1091) as compared to the expression of antigen-specific antibodies of B cells.
  • this study demonstrates that gp39 + T cells and cytokine-PC are simultaneously upregulated in vivo after immunization. Furthermore, the observed high frequencies of gp39 + cells and IL-4-PC in close conjunction to antigen-specific B cells, may represent Th2-B cell interactions which propagate the preferential development of IgG1 + AFC in the antibody response against KLH. Finally, the data suggest that the initial cognate B cell activation and the subsequent regulation of B cell differentiation by T cells occur in the non-follicular areas of the spleen, namely the outer-periarteriolar sheaths and around the terminal arterioles.
  • anti-gp39 treatment did not cause T h deletion or anergy.
  • proliferation of antigen-primed lymph node cells in vitro was not inhibited by anti-gp39.
  • Anti-gp39 may mediate its profound immunosuppressive effects on humoral immunity by blocking gp39-CD40 interactions.
  • these studies establish gp39-CD40 as an important receptor-ligand pair for the targeting of therapeutic antibodies to control TD humoral responses.
  • mice Female, 6-8 week old BALB/C mice (Jackson Laboratories, Bar Harbor, Me.) were used for the in vivo experiments presented in this study. Animals were maintained in the specific pathogen-free animal facility at Dartmouth Medical School.
  • T h 1 an I-A d -restricted, rabbit Ig-specific T h 1 clone Kurt-Jones et al. (1987) J Exp. Med. 166:1774 was obtained from Dr. David Parker, University of Mass. at Worcester.
  • D1.6 will be referred to as T h 1.
  • MR1 hamster anti-murine gp39 mAb (14) was purified by DEAE HPLC from ascites fluid.
  • Hamster Ig HIg
  • RG7/7.6.HL a mouse anti-rat ⁇ chain (strongly crossreactive with hamster ⁇ chain) antibody, (RG7), (Springer et al. (1982) Hybrid. 1:257) was conjugated with HRPO or FITC and used as a secondary reagent to detect MR1 and HIg.
  • Affinity-purified goat anti-mouse IgM, IgG 1 , IgG 2a , IgG 2b and IgG 3 were used as detection antibodies in the antigen specific ELISAs as well as in the total IgM and IgG1 ELISAs.
  • Chimeric-L6 (Chi-L6), a humanized IgG 1 specific for the tumor antigen L6 (21), was kindly provided by Bristol-Myers Squibb Pharmaceutical Research Institute, Seattle Wash. Anti-CD4, GK 1.5 (Wilde et al. (1983) J. Immunol. 131:2178) was prepared by HPLC purification of ascites fluid.
  • Sheep red blood cells (SRBC) were purchased from Colorado Serum Co. (Denver, Colo.).
  • Sea Plaque agarose for use in anti-SRBC plaque assay was obtained from FMC Corporation (Rockland, Mass.).
  • Baby rabbit complement was purchased from Cedarlane (Hornby, Ontario, Canada).
  • mice were immunized with 200 ⁇ l of 1% SRBC or TNP-SRBC suspension (i.v.).
  • the IgM, anti-SRBC response was assayed 5 d after administration of antigen using a modification of the Jerne plaque assay (Jerne et al. (1974) Transplant Rev. 18:130).
  • IgM anti-TNP responses were measured by ELISA on day 6.
  • Primary responses to the heterologous immunogobulin Chi-L6 were generated by i.p. immunization of 100 ⁇ g Chi-L6, in alum, per mouse.
  • the serum IgM anti-Chi-L6 antibody response was measured after 7 d.
  • Primary responses to TNP-Ficoll were generated by immunization with 25 ⁇ g of TNP-Ficoll i.p.
  • the IgM anti-TNP response was measured on day 6 by ELISA.
  • mice were immunized with KLH in CFA (50 ⁇ g; i.p.). Mice were subsequently challenged with 10 ⁇ g of soluble KLH (i.p.) three months later.
  • the anti-KLH antibody response was measured on d7 from the serum of immune mice utilizing isotype specific ELISAs.
  • Secondary antibody responses to Chi-L6 were generated by challenging Chi-L6 immune mice with 10 ⁇ g soluble Chi-L6 i.p.
  • the serum IgG, anti-Chi-L6 antibody response was measured after 7 d.
  • the antigen specific IgM, IgG 1 , IgG 2a , IgG 2b , IgG 3 , and IgE antibody titers were determined using isotype specific ELISAs. Briefly, antigen, (1 mg/ml of KLH, Chi-L6, TNP 16 -BSA, or TNP 2 -BSA in PBS) was absorbed onto flexible polyvinyl microtiter dishes, overnight at 4° C. Plates were washed and blocked with PBS-1% FCS-azide. Diluted serum samples were incubated for 2 hours at 37° C.
  • Units represent arbitrary values based on the titration curve of a standard immune serum. All experimental groups were titered from 1:100 to 1:100,000 and the titer ascertained based on multiple point analysis. The levels of anti-KLH, anti-Chi-L6 and anti-TNP antibodies in unchallenged controls were below detection.
  • the serum was run on a 7.5% SDS gel under non-reducing conditions, transferred to nitrocellulose, and blotted with HRPO-conjugated RG7. Following chemiluminescent detection, areas of the blot corresponding to 150-165 kDa were scanned and digitized using an Apple Scanner and the Image 4.1 software program.
  • Anti-CD3-activated T h 1, which express gp39, were stained with dilutions of serum from mice receiving 750 ⁇ g anti-gp39 (250 ⁇ g on d0, d2, d4) to determine the amount of biologically active gp39 remaining in the serum. Titrations of serum containing anti-gp39 were incubated with activated T h 1 cell clones for 30 minutes at 4° C., followed by washing and subsequent incubation with FITC-RG7, 30 minutes at 4° C. A standard curve of MFI vs anti-gp39 concentration was generated using purified anti-gp39.
  • mice were immunized by footpad injection with 50 ⁇ l of KLH in CFA.
  • Popliteal lymph nodes were removed on d5 after the administration of antigen and single cell suspensions were prepared.
  • Lymph node cells were cultured in 200 ⁇ l complete RPMI at 100,000 cells per well in 96-well microtiter plates in the presence or absence of 100 ⁇ g/ml KLH.
  • Anti-gp39 (MRI) was added to the cultures as indicated. Cellular proliferation was assessed using 3 H-thymidine incorporation 3 d after initiation of culture.
  • mice were immunized with SRBC (200 ⁇ l of 1% SRBC, i.v.) and administered anti-gp39 or HIg (250 ⁇ g on d0, d2, d4).
  • SRBC 200 ⁇ l of 1% SRBC, i.v.
  • HIg 250 ⁇ g on d0, d2, d4
  • mice were immunized with SRBC (200 ⁇ l of 1% SRBC, i.v.) and administered anti-gp39 or HIg (250 ⁇ g on d0, d2, d4).
  • SRBC 200 ⁇ l of 1% SRBC, i.v.
  • HIg 250 ⁇ g on d0, d2, d4
  • mice were immunized with SRBC (200 ⁇ l of 1% SRBC, i.v.) and administered anti-gp39 or HIg (250 ⁇ g on d0, d2, d4).
  • mice were immunized with TNP-SRBC (200 ⁇ l of 1% TNP-SRBC i.v.) Serum IgG 1 anti-TNP titers were ascertained on d6 post-transfer.
  • Anti-gp39 Inhibits the Generation of Primary Antibody Responses to Erythrocyte Antigens
  • mice On d5, the primary anti-SRBC antibody response of anti-gp39-treated, HIg-treated and control mice was ascertained.
  • administration of as little as 300 ⁇ g/mouse (100 ⁇ g/mouse on d0, d2, and d4) of anti-gp39 reduced the anti-SRBC primary immune response by 66%. Results from these experiments demonstrate that anti-gp39 treatment ablates primary antibody responses in vivo.
  • mice immunized with SRBC were treated with anti-gp39 for 4 d and assayed at various later time points for the capacity to mount a primary anti-SRBC response.
  • all animals were immunized with SRBC on do and administered anti-gp39 or HIg on d0, d2, d4.
  • the IgM anti-SRBC PFC response was measured for one group on d5. Additional SRBC-immune groups were challenged with SRBC on d7 or d14.
  • FIG. 15 B Five days following each antigenic challenge (d12 and d19, respectively), the IgM anti-SRBC PFC response was measured. The results of one such experiment are depicted in FIG. 15 B. As in FIG. 15A, the primary anti-SRBC responses were inhibited 80-90% 5 d after anti-gp39 administration was begun. In addition, the primary anti-SRBC responses 12 d and 19 d following anti-gp39 treatment were also inhibited >90%. These results demonstrate that even brief anti-gp39 treatment results in prolonged inhibition of primary antibody responses.
  • mice were challenged with soluble KLH (10 ⁇ g/mouse/i.v.).
  • d0 mice were also given 250 ⁇ g of anti-gp39 or HIg, followed by anti-gp39 or HIg on d2 and d4.
  • d7 FIG. 16B
  • d14 FIG.
  • mice were bled and the titers of IgM, IgG 1 , IgG 2a , IgG 2b , IgG 3 and IgE anti-KLH were determined.
  • the results demonstrate several points: 1) challenge with soluble KLH induced an enduring secondary immune 35 response that persisted for up to 14 d; 2) the administration of anti-gp39 significantly reduced the secondary anti-KLH response of the isotypes measured when compared to the administration of equal quantities of HIg; and 3) the immunosuppressive effects of anti-gp39 appeared to be sustained for at least 14 d after the initiation of anti-gp39 treatment.
  • results from these experiments demonstrate that similar to primary humoral immune responses, the generation of secondary humoral immune responses were also blocked by anti-gp39.
  • FIGS. 15A and B demonstrate the immunosuppressive activity of anti-gp39 during a primary response to a strongly immunogenic particulate antigen, SRBC.
  • SRBC strongly immunogenic particulate antigen
  • FIGS. 17A-17B depicts the results of one such experiment.
  • the primary antibody response to Chi-L6 in mice treated with anti-gp39 is inhibited by >90% when compared to HIg-treated mice.
  • the secondary, IgG, response to Chi-L6 is similarly inhibited.
  • FIG. 18 A and Panel A demonstrates that animals immunized with the TD antigen TNP-SRBC elicit significant anti-TNP serum antibody responses.
  • anti-gp39 treatment dramatically inhibits the primary anti-TNP response generated in these mice.
  • mice immunized with TNP-Ficoll mount a higher titered anti-TNP antibody response (FIG. 18 B); however, treatment with anti-gp39 does not inhibit the antibody response to TNP-Ficoll.
  • results from these experiments demonstrate that, unlike responses to TD antigens, anti-gp39 does not block the generation of humoral responses to TNP-Ficoll, suggesting that responses to TI antigens may be gp39-independent.
  • Immune suppression by anti-gp39 may be mediated by: 1) the negative signalling of gp39-bearing T cells causing T h anergy; 2) mAb-mediated cytotoxic deletion of anti-gp39 beating CD4+ T cells; and/or 3) the blocking of gp39 binding to CD40.
  • a series of experiments were performed to gain insights into which of these mechanisms may be operative in the protracted immune suppression observed with anti-gp39 therapy.
  • antigen-specific T h were deleted or energized by anti-gp39 therapy, antigen-specific T h function from gp39-treated mice was measured by adoptive transfer.
  • mice were immunized with SRBC (to prime SRBC-specific T h ) and administered anti-gp39 or HIg (250 ⁇ g/mouse on d0, d2, d4).
  • SRBC-immune spleen cells from HIg-treated or anti-gp39-treated mice were adoptively transferred into recipient mice with TNP-immune spleen cells as a source of TNP-primed B cells.
  • Mice were simultaneously challenged with TNP-SRBC, and the IgG 1 anti-TNP titer ascertained on d5. As shown in FIG.
  • SRBC helper activity from HIg-treated and anti-gp39-treated mice were similar, indicating that anti-gp39 treatment did not alter T h function or block the priming of T h .
  • antigen-responsive T h were not deleted or energized as a result of anti-gp39 treatment, as they provided helper-effector function upon transfer.
  • mice were primed in the footpads with KLH in CFA (50 ⁇ g/footpad). After 5 d, the popliteal lymph nodes were removed and lymph node cells were cultured in vitro with antigen in the presence or absence of anti-gp39. The results demonstrate that the addition of anti-gp39 to cultures of KLH-primed lymph node cells does not block the antigen-induced proliferative response.
  • lymph node cells cultured with antigen alone were similar to that of cells cultured with antigen plus as much as 25 ⁇ g/ml of anti-gp39.
  • in vivo primed lymph node cells cultured in vitro with 100 ⁇ g/ml KLH incorporated 87854 ⁇ 3522 cpm of 3 H-thymidine whereas cells cultured with antigen and 25 ⁇ g/ml of anti-gp39 incorporated 89084 ⁇ 1619 cpm. Cells cultured with no antigen yielded 2819 ⁇ 453 cpm.
  • the present study demonstrates that an anti-gp39 antibody, which blocks gp39-CD40 interactions in vitro, results in profound inhibition of both primary and secondary humoral immune responses to TD antigens, but not TI-type II antigens.
  • anti-gp39 treatment does not block the priming of, or proliferation of, antigen-primed T h cells. Therefore, the gp39-CD40 ligand-receptor pair can be used as a target for the therapeutic manipulation of the humoral immune response.
  • mice with anti-gp39 inhibited the primary immune response to SRBC and heterologous Ig>90% for prolonged periods of time. Assuming that anti-gp39 is mediating the inhibition by blocking gp39 function, these data implicate gp39-CD40 interactions as essential in the development of primary immune responses to TD antigens. Immunohistochemical analysis establish that gp39 is induced as a consequence of immunization with TD antigens and may be of functional significance.
  • the in situ studies of gp39 expression illustrate that the initial site of gp39-CD40 interactions during primary humoral immune responses is in the peripheral aspects of the periarteriolar lymphoid sheaths (PALS) and around the terminal arterioles (TA) of the spleen. It is at these sites that conjugates between gp39-expressing T h and antigen-specific B cells were found juxtaposed, suggesting that the outer PALS is a major site of T cell-B cell interactions during primary humoral immune responses. Therefore, the PALS may be the site at which anti-gp39 interacts with gp39-expressing T h cells to ultimately inhibit T-B interaction and subsequent Ig production.
  • PALS periarteriolar lymphoid sheaths
  • TA terminal arterioles
  • mice primed to KLH in CFA were also shown to be inhibited by the administration of anti-gp39. Consistent with the reduction of anti-SRBC PFC by anti-gp39, reductions in serum antibodies titers to antigenic challenge were also observed.
  • the serum titers of all anti-KLH Ig isotypes measured (IgM, IgG 1 , IgG 2a , IgG 2b , IgG 3 , and IgE) were reduced by the treatment of mice with anti-gp39.
  • the effect of anti-gp39 administration was apparent for at least 14 d after secondary challenge with antigen, establishing a persistent immune suppression by anti-gp39.
  • Anti-gp39-mediated immune suppression of secondary responses to KLH is not unique to KLH, since secondary immune responses to heterologous Ig and heterologous erythrocytes were also inhibited by anti-gp39 therapy.
  • the anatomical distribution of gp39-expressing T h was identical to that observed upon primary immunization, however, the frequency of gp39-expressing T h in immune spleen was increased over that observed during primary immune responses. No gp39-expressing T h were found in the germinal centers or follicles of immune spleen. Thus, it appears that B cells are triggered to respond to activated T h cells in the PALS and TA of the spleen and later migrate to the follicles and germinal centers.
  • the signal transduction pathway initiated in B cells as a consequence of interacting with activated T h is examined.
  • plasma membranes isolated from activated T h did not trigger an increase in the B cell intracellular concentrations of cAMP or calcium.
  • PM Act did not stimulate PKC activation as measured by MARCKS phosphorylation and PKC translocation.
  • PM Act induced the tyrosine phosphorylation of several B cell substrates, including a 43 kd protein which comigrated with ERK 1.
  • Neutralizaing antibodies directed against gp39 blocked PM Act -induced B cell PTK activity; conversely, agonistic antibodies directed against CD40 stimulated B cell PTK activity. Since triggering through CD40 appear essential for T h -dependent B cell activation, these results suggest that CD40 initiates a signal transduction pathway in B cells which is different from the pathway initiated by mIg or MHC class II.
  • mice Female DBA/2j mice (Jackson Laboratories, Bar Harbor, Me.) at 6-8 weeks of age were used for the preparation of filler cells to support the growth of T h clones of plasma membrane isolation and in the preparation of resting B lymphocytes.
  • T h 1 were cultured at 8 ⁇ 10 6 cells/well in cluster wells (6 well, Corning Glass Works, Corning N.Y.) coated with 10 ⁇ g/ml of anti-CD3 in PBS (Leo et al. (1987) Proc. Natl. Acad. Sci. USA 84:1374). Cells were incubated for 16 hours at 37° C., harvested, and washed.
  • Plasma membranes were prepared by discontinuous sucrose gradient sedimentation, as described previously (Noelle et al. (1991) J. Immunol. 146:1118).
  • B cells were prepared from the spleens of 6-8 week old DBA/2J mice (Jackson Laboratories), as described in (Noelle et al. (1991) J. Immunol. 146:1118).
  • mice rIL4 10 ng/ml; kindly provided by Drs. C. Maliszewski and K. Grabstein, Immunex Corporation, Seattle, Wash.
  • RPMI RPMI containing 10% FCS (Hyclone, Logan, Utah), for 16 hours at 37° C.
  • anti-CD3 145-2C11 (Leo et al. (1987) Proc. Natl. Acad. Sci. USA 84:1374)
  • anti-CD4 RL172/4 (Mizouchi et al. (1989) J. Immunol. 142:270) and GK1.5 (Armitage et al. (1992) Nature 357:80)
  • anti-MHC class II M5/114.15.2 (Bhattacharya et al. (1981) J. Immunol 127:2488); anti-gp39 (MR1) (Noelle et al. (1992) Immunol. Tod. 13:431); and anti-TcR ( ⁇ / ⁇ ): H57-597 (Kubo et al. (1989) J. Immunol. 142:2736).
  • E ⁇ rosetted B cells were isolated from blood samples obtained from normal donors. Sheep red blood cells (RBCs; Kroy Medical Inc., Stillwater, Minn.) were incubated in AET (2-aminoethyl isothiouronium bromide, 140 mM, pH 9.0; Sigma Chemical Co.) for 15 minutes at 37° C. and washed. Peripheral blood lymphocytes (PBLs) were separated from whole blood by flotation on Ficoll gradients and rotation to remove monocytes. PBLs were then mixed with AET-treated sheep RBCs, at a ratio of 1 PBL:30 sheep RBCs, for 15 minutes at 37° C.
  • AET Peripheral blood lymphocytes
  • Murine resting B cells at 10 6 cells/sample were incubated with PM rest (1 ng-10 ⁇ g/sample), PM Act (1 ng-10 ⁇ g/sample), anti-MHC class II (50 ⁇ g/ml) or forskolin (50 ⁇ M; Sigma Chemical Co.) in RPMI containing 10% FCS (Hyclone) and rolipram (0.1 mM; Diagnostic Products Corp.) for various time points and volumes.
  • the reaction was stopped with the addition of cold TCA (2 mM) and samples were analyzed with a 3 H-cAMP Assay Kit, (Diagnostic Products Corp.). Briefly, samples were ether-extracted 3 times, dried, and resuspended in Tris-EDTA.
  • Murine resting B cells at 10 7 cells/ml were loaded with Indo-1 (5 ⁇ M; Molecular Probes Inc.), in RPMI containing 10% FCS (Hyclone), for 30 minutes at 37° C. Cells were washed, aliquoted at 10 6 /sample, and maintained at 37° C. Indo-1 fluorescence was measured on an Ortho Systems 50H Cytofluorograph (Ortho Pharmaceutical, Raritan, N.J.).
  • Murine resting B cells at 10 7 cells/ml were labeled with 32 P-orthophosphate (0.5 mCi/ml; New England Nuclear, Dupont Co.) for 1 hour at 37° C. in phosphate-free DMEM containing 10% FCS (Hyclone).
  • Extraction Buffer (10 mM Tris, pH 7.4, 0.15 mM NaCl, 0.02% NaN 3 , 0.5% NP-40, and 100 ⁇ g/ml each of aprotinin, leupeptin, pepstatin A (Sigma Chemical Co.)) and immunoprecipitated for 16 hours at 4° C. with an anti-MARCKS polyclonal antiserum followed by Protein A Sepharose for 1 hour at 4° C. (Sigma Chemical Co.).
  • the anti-MARCKS polyclonal antiserum was generated through the use of a MARCKS peptide, kindly provided by Dr. P. Hornbeck, University of Maryland, Baltimore, Md.
  • Murine resting B cells at 5 ⁇ 10 5 cells/sample were incubated with pM rest (1 ng-10 ⁇ g/sample), PM Act (1 ng-10 ⁇ g/sample), PMA (100 ng/ml; Sigma Chemical Co.) or G ⁇ M IgG F(ab′)2 (50 ⁇ g/ml; Cappel, Organon Teknika, Corp.) in RPMI, containing 10% FCS (Hyclone), for various time points at 37° C. and volumes. Cells were washed in PBS and digitonin permeabilized according to Berry et al. (Berry et al. (1989) J. Immunol. 143:1407).
  • cells were resuspended in Translocation Buffer (20 mM MOPS (Sigma Chemical Co.) pH 7.2, 10 mM EGTA (Sigma Chemical Co.), 5 mM EDTA (Fisher Chemical Co., Fair Lawn, N.J.), 0.5 mg/ml digitonin (Wako Chemicals USA, Inc.), 100 ⁇ g/ml each of pepstatin A, leupeptin, and aprotinin (Sigma Chemical Co.)) set on ice for 5 minutes and spun at 300 rpm for 2 minutes.
  • Translocation Buffer 20 mM MOPS (Sigma Chemical Co.) pH 7.2, 10 mM EGTA (Sigma Chemical Co.), 5 mM EDTA (Fisher Chemical Co., Fair Lawn, N.J.), 0.5 mg/ml digitonin (Wako Chemicals USA, Inc.), 100 ⁇ g/ml each of pepstatin A, leupeptin, and aprotinin (Sigma
  • the supernatant (cytosolic fraction) and the remaining pellet (membrane fraction) for each sample were analyzed via SDS/Page electrophoresis and Western blotting; samples were electrophoresed on a 7.5% SDS/Page gel and transferred to 0.2 ⁇ m nitrocellulose (Schleicher & Schuell).
  • the blot was probed with an anti-PKC polyclonal antiserum (Kraft et al. (1988), J. Biol. Chem. 264:8437), kindly provided by Dr. C. Ashendel, Purdue University, Purdue, Ind., followed by GaCh IG-HPRO (Southern Biotechnology Inc.) and developed with the ECL Western Detection Blotting System (Amersham Co., Arlington Heights, Ill.).
  • B cells at 5 ⁇ 10 5 cells/100 ⁇ l were warmed in 96-well plates (Costar) with RPMI containing 10% FCS (Hyclone) for 30 minutes at 4° C. or 37° C.
  • the following activators were added to the B cell cultures and incubated for various time points at 4° C. or 37° C.: PM rest (1 ng-10 ⁇ g/10 ⁇ l), PM Act (1 ng-10 ⁇ g/100 ⁇ l), G ⁇ M IgG F(ab′)2 (50 ⁇ g/ml; Cappel, Organon Teknika, Corp.), anti-CD40 (1 ⁇ g/ml; G28.5; kindly provided by Dr. J.
  • samples were electrophoresed on a 10% SDS/Page gel and transferred to 0.45 ⁇ m PVDF Immobilon (Millipore, Bedford, Mass.).
  • the blot was probed with the anti-phosphotyrosine antibody 4G10 (Davis et al. (1991), Science 252:712), kindly provided by Dr. R. Frackelton, Brown University, Buffalo R.I., followed by G ⁇ M IgG 2b -HPRO (Southern Biotechnology Inc.) and then developed with the ECL Western Detection Blotting System (Amersham Co.).
  • Murine resting B cells at 5 ⁇ 10 5 cells/sample were lysed in Extraction Buffer, prepared for SDS/Page electrophoresis and Western blotting as described above. Blots were then probed with anti-ERK polyclonal antiserum #691 (Boulton et al. (1991) Cell. Regulat. 2:357), kindly provided by Dr. M. Cobb, Southwestern Medical Center, Dallas, Tex., followed by G ⁇ Rb IgG-HPRO (Southern Biotechnology Inc.) and developed with ECL Western Detection Blotting System (Amersham Co.).
  • phosphotyrosine blots were stripped of all detecting antibodies by incubating blots in 0.2M glycine and 0.05% Tween 20 (Sigma Chemical Co.), pH 2.5, for 2 hours at 80° C. Blots were then probed with anti-ERK polyclonal antiserum #691, as described above.
  • B cells were labeled with 32 porthophosphate and incubated with PM rest , PM Act or phorbol myristate acetate (PMA).
  • Samples were immunoprecipitated with an anti-MARCKS polyclonal antiserum (Hornbeck et al. (1989) Molec. Cell. Biol. 9:3727) and prepared for electrophoresis and autoradiogaphy as described in Materials and Methods.
  • the MARCKS protein has a molecular weight of approximately 80 kd.
  • PMA which directly activates PKC (Schmidt et al. (1975) Cancer Res.
  • PMA and anti-mIg included as positive controls, induced significant increases in PKC translocation to the B cell membrane at 5 minutes post-culture; at this time point, B cells alone exhibited a basal level of PKC translocation (FIG. 25 ).
  • anti-mIg induced the translocation of two PKC isoforms at approximately 80 kd and 78 kd (FIG. 25 ).
  • PM Act did not induce a detectable increase in PKC translocation at 30 minutes post-culture as compared to PM rest (FIG. 25 ).
  • Analysis of cytosolic fractions revealed a concomitant loss of PKC in response to PMA or anti-mIg, but not to PM rest or PM Act .
  • Additional experiments were performed with various PM Act concentrations, incubation times and IL4 pretreated B cells (Table II). Despite these manipulations, PM Act did not induce detectable increases in PKC translocation to the B cell membrane.
  • FIGS. 26A-26B The increased number and intensity of phosphotyrosine proteins shown in FIGS. 26A-26B is most likely the result of PM Act stimulating B cell PTK activity. However, it is possible that these proteins are passively acquired by the B cell as a result of binding PM Act which contain an array of phosphoproteins. To eliminate this possibility, a number of studies were performed. First, if the phosphoproteins were constitutively expressed by PM Act , passive acquisition of the phosphoproteins should be observed at both 4° C. and 37° C. As such, B cells were incubated with either PM rest or PM Act at 4° C. or 37° C. and prepared for phosphotyrosine analysis, as described previously.
  • PM Act enhanced the tyrosine phosphorylation of several proteins, in a concentration dependent manner, at 37° C. but not at 4° C. (FIG. 27 A). Although there may appear to be some differences in the PM Act -induced phosphotyrosine profile, as compared to FIG. 26B, the phosphotyrosine profile in FIG. 27A was underexposed in order to demonstrate clearly the concentration dependence of the PM Act effect. These results suggest that tyrosine phosphorylation was not a passive binding event of PM Act , but rather an active event in the B cell. In order to verify that PM Act bound to B cells at 4° C., B cells were cultured with PM Act at both 4° C. and 37° C.
  • CD40 is homologous to the low affinity nerve growth factor receptor (p75 NGFR ) (Stamenkovic et al. (1989) The EMBO J 8:1403), which together with the trkA gene product (p140 trkA ), initiates a protein tyrosine kinase (PTK) pathway (Miyasaka et al. (1991) Proc. Natl. Acad. Sci. USA 88:2653); and antibody-mediated cross-linking of CD40 induces the tyrosine phosphorylation of several B cell substrates (Uckun et al.
  • PTK protein tyrosine kinase
  • Human B cells were utilized to identify CD40 as the trigger molecule for the enhanced tyrosine kinase activity. Human B cells were incubated with anti-CD40 (Clark et al. (1986) Proc. Natl. Acad. Sci. 83:4494), mIgG, or PMA plus ionomycin, for various time points and prepared for phosphotyrosine analysis as described previously. PMA plus ionomycin, which induces the tyrosine phosphorylation of B cell substrates (Uckun et al. (1991) J. Biol. Chem. 266:17478), was included as a positive control; mIgG was included as a negative control for anti-CD40.
  • T h triggers a tyrosine kinase-dependent signalling pathway in B cells.
  • activated T h enhanced the tyrosine phosphorylation of several proteins, including a 43 kd protein which comigrated with ERK I, in the absence of increased calcium mobilization and protein kinase C (PKC) activation.
  • PKC protein kinase C
  • an anti-gp39 monoclonal antibody which inhibited T h -dependent B cell activation, blocked increased tyrosine phosphorylation suggesting that gp39-CD40 interactions triggered signal transduction between T h and B cells.
  • CD40 was further implicated as a trigger molecule since an agonistic anti-CD40 monoclonal antibody enhanced tyrosine phosphorylation of several human B cell substrates, including a 43 kd protein.
  • Antibody mediated cross-linking of either mIg or MHC class II has facilitated the study of second messenger events initiated during B cell activation. These studies have shown that cross-linking of mIg or MHC class II triggers increased intracellular cAMP, production, PTK activity, phosphoinositol turnover, intracellular calcium mobilization and PKC activation. Additionally, Lane et al. have reported increased phosphoinositol turnover and intracellular calcium concentration in a human B lymphoblastoid cell line which present peptide to an antigen-specific T cell clone (Lane et al. (1991) J. Immunol. 147:4103).
  • B cells are activated by both thymus-independent (TI) or thymus-dependent (TD) antigens. Although both of these antigenic types initiate B cell proliferation and antibody production, they do so through different triggering systems. TI antigens deliver mitogenic signals via mIg; whereas, TD antigens ultimately trigger B cell activation via CD40. It appears, based on the data presented in this section, that the biochemical signalling pathways employed to initiate B cell activation by TI and TD antigens are quite divergent, and that unlike TI antigens, TD antigens, with T cell help, trigger signal transduction through CD40, initiating PTK activity in the absence of increased intracellular cAMP production, intracellular calcium mobilization and PKC activation.
  • mice DBA/2j mice were used and obtained from NCl laboratories (Bethesda, Md.).
  • Chi-L6 was precipitated on alum (aluminum potassium sulfate, Sigma, St. Louis, Mo.) by adding 10% alum solution to 1 mg/ml Chi-L6 to a ratio of 1:1(v:v). The pH was then adjusted to 6.5 with NaOH and then the reaction mixture was allowed to stand for 30 minutes at room temperature. The mixture was then washed twice with PBS (pH 7.4) and then resuspended at 1 mg/ml of antigen.
  • alum aluminum potassium sulfate, Sigma, St. Louis, Mo.
  • mice Three groups of mice, with 3 in each group, were immunized with 100 ⁇ g i.p. of Chi-L6 on alum. One group then received 250 mg injections i.p. of the anti-gp39 antibody, MR1, one days 0, 2, 4, and 6. The second group received the same injection regime but using an irrelevant hamster control antibody (HIg) and the third group received no antibody treatment. All animals were bled before the primary immunization and then bled after 1 week. Serum samples were then prepared to analyze for the presence of anti-chi-L6 antibodies (IgM) in the primary immune response. After this period mice were injected with 100 ⁇ g of soluble chi-L6 i.p. and no further antibody treatment was given. These mice were then left for 2 weeks, when they were again bled and the serum samples prepared for detection of IgG antibodies to chi-L6, indicative of a secondary immune response.
  • IgM anti-chi-L6 antibodies
  • An ELISA was developed to detect the presence of anti-Chi-L6 antibodies in the serum of the mice.
  • the wells of a 96 well polyvinyl microtitre plate were coated with Chi-L6 at a concentration of 10 ⁇ g/ml (100 ⁇ l), and incubated overnight at 4° C.
  • the plate was then washed 3 times and 100 ⁇ l of PBS containing 5% FCS and 0.02% azide was added to each well as the blocking step and incubated for 60 minutes at 37° C.
  • the plate was again washed 3 times and the serum samples were then added at various dilutions.
  • the test serum and standard serum were incubated for 2 hours at 37° C. and then removed by washing the plate 3 times.
  • alkaline phosphatase conjugate antibody (goat anti-mouse IgG 1 or IgM, (1:500 dilution), Southern Biotechnology Inc. Birmingham, Ala.) was added to each well and after 2 hours of a 37° C. incubation the conjugated antibody was removed by thorough washing.
  • FIG. 32 indicates that the treatment of immunized animals with anti-gp39 antibody does in fact inhibit the primary IgM response to Chi-L6.
  • the profound induction of anti-chi-L6 antibodies is markedly reduced by the MR1 treatment.
  • animals are then challenged with the soluble form of Chi-L6 but receive no further antibody treatment, it is found that the IgG response, indicative of a secondary immune response, is also inhibited.
  • gp39-CD40 interactions The role of gp39-CD40 interactions in the development of autoimmune disease has been investigated in vivo using an antibody that blocks gp39-CD40 interactions.
  • Arthritis induced in mice by immunization with type II collagen (CII) was completely inhibited by the administration of anti-gp39.
  • Administration of anti-gp39 to CII-immunized mice blocked the development of joint inflammation, serum antibody titers to collagen, the infiltration of inflammatory cells into the subsynovial tissue and erosion of cartilage and bone.
  • HIM hyper IgM syndrome
  • Ig immunoglobulin
  • gp39 proteins from HIM patients cannot trigger B cell activation and are unable to bind to CD40 (Korthauer et al. (1993) Nature 361:539).
  • TD thymus dependent
  • a monoclonal antibody (mAb) specific to murine gp39, MR1 blocks the binding of gp39 to CD40 and also blocks T helper cell (Th)-dependent B cell activation in vitro.
  • Th T helper cell
  • CIA Collagen type II-induced arthritis
  • CII heterologous native type II collagen
  • mice three groups of mice (eight mice per group) were immunized with CII in CFA and then one week later each group received either no mAb, anti-gp39, or irrelevant hamster Ig (HIg). Mice were subsequently administered antibody (250 ⁇ g per mouse) every four days until the end of the experiment. This antibody treatment regime was used because previous titrations established that this regime of mAb administration inhibited greater that 90% of the primary (IgM) anti-SRBC response (Courtenay et al. (1980) Nature 283:665). Antibody half life is estimated to be 12 days and thus injections every four days maintains adequate levels for the blocking of gp39 function.
  • Another consequence of immunization with CII is a precipitous rise in serum IgG titers to CII.
  • the serum IgG anti-CII titers were inhibited compared to the anti-CII titers found in mice untreated or treated with HIg (FIG. 35 ).
  • titers of anti-CII in anti-gp39-treated mice were similar to titers found in nonimmune control sera (Cooper et al. (1992) Clin. Exp. Immunol. 89:244-250).
  • mice with GVHD there was a marked increased in the spleen size that was reversed by the administration of anti-gp39. Both spleen size and reduced Ig levels remained normal for extended time periods when the antibody treatment was terminated.
  • DBA/2 (H-2 d ) and B6D2F 1 ((C57BL/6 (H-2 d ) ⁇ DBA/2)F 1 hybrid) mice were obtained from the NCI laboratories (Bethesda, Md.). All the mice used in this study were female.
  • Chronic GVHD was induced by the i.v. injection of parental (DBA/2) spleen cells into non-irradiated (C57BL/6 ⁇ DBA/2) F 1 hybrid recipients (Fast (1990) J. Immunol. 144:4177).
  • Parental mice were anesthetized and killed by cervical dislocation in preparation for removal of the spleen.
  • Dissociated spleen cells were washed and resuspended in RPMI 1640 medium (Whittaker, Walkersville, Md.) for i.v. injection into the F 1 recipients.
  • MR1 was produced in our laboratory as previously described (Noelle et al. (1992) Proc. Natl. Acad. Sci. USA 89:6550). All anti-IgG 1 and IgA antibodies and standard controls were obtained from Southern Biotechnology Associates, Inc., (Birmingham, Ala.).
  • mice with ongoing GVHD were treated with MR1 following the regime indicated in FIG. 36 .
  • GVHD induction was determined by splenomegaly and hyperproduction of Ig in vitro. Spleens were removed from treated or untreated mice with ongoing GVHD. Cells suspensions were treated with Tris-buffered ammonium chloride and total white blood cell counts were determined by visual hemocytometer counting. Cells were incubated (5 ⁇ 10 6 ) in 1 ml of RPMI-1640 medium (supplemented with 10% fetal calf serum, 25 mM HEPES, 2 mM L-glutamine, 5000 U/ml penicillin and 5000 ⁇ g/ml streptomycin) for 3 days at 37° C., 5% CO 2 .
  • Cell supernatants were collected by pelleting the cells and Ig was quantified by an isotype-specific ELISA assay.
  • the ELISA was performed by absorbing goat anti-mouse IgG 1 or IgA (10 ⁇ g/ml; Southern Biotechnology Associates, Inc., Birmingham, Ala.) in PBS onto wells of a 96-well polyvinyl microtitre plate for 1 hour at 37° C. then overnight at 4° C. Plates were washed and blocked with PBS containing 1% FCS for 1 hour at 37° C.
  • FIGS. 38A-38B indicates that mice induced with GVHD for 1 week and 2 weeks resulted in spleens with almost twice the cell numbers compared to normal F 1 recipients.
  • mice When mice were treated with the anti-gp39 antibody (250 ⁇ g/mouse, days 0,2,4, and 6), the cell numbers returned to levels of the normal spleens and remained low for 1 week after the antibody treatment was terminated (2 wk GVHD+1 wk MR1 (FIG. 37 )).
  • mice with GVHD were administered anti-gp39.
  • spleens were removed from control and treated mice.
  • IgG 1 and IgA were assayed for the presence of IgG 1 and IgA.
  • Spleens from mice with GVHD produced high levels of IgA and IgG 1 (FIGS. 38 A and B).
  • GVHD mice were treated with anti-gp39 for 1 week, the levels of all IgG 1 and IgA returned to basal levels. The levels of both IgG 1 and IgA remained at basal levels for 1 week after termination of anti-gp39.

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